Immunogenic compositions for the prevention and treatment of meningococcal disease

ABSTRACT

The present invention relates to  Neisseria  ORF2086 proteins, crossreactive immunogenic proteins which can be isolated from nesserial strains or prepared recombinantly, including immunogenic portions thereof, biological equivalents thereof, antibodies that immunospecifically bind to the foregoing and nucleic acid sequences encoding each of the foregoing, as well as the use of same in immunogenic compositions that are effective against infection by  Neisseria meningitidis  serogroup B.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/455,326, filed Apr. 25, 2012, which is a continuation of U.S.application Ser. No. 13/333,123, filed Dec. 21, 2011, now U.S. Pat. No.8,563,007, which is a divisional of U.S. application Ser. No.10/492,427, filed Oct. 7, 2004, now U.S. Pat. No. 8,101,194, which is aNational Stage of International Application No. PCT/US02/32369, filedOct. 11, 2002, which claims the benefit of U.S. Provisional ApplicationNo. 60/328,101, filed Oct. 11, 2001, and U.S. Provisional ApplicationNo. 60/406,934, filed Aug. 30, 2002, all of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to Neisseria ORF2086 proteins (Subfamily Aand Subfamily B), which may be isolated from bacterial strains such asthose of Neisseria species, including strains of Neisseria meningitidis(serogroups A, B, C, D, W-135, X, Y, Z and 29E), Neisseria gonorrhoeae,and Neisseria lactamica, as well as immunogenic portions and/orbiological equivalents of said proteins. The present invention alsorelates to antibodies that immunospecifically bind to said proteins,immunogenic portions and/or biological equivalents. Further, the presentinvention relates to isolated polynucleotides comprising nucleic acidsequences encoding any of the foregoing proteins, immunogenic portions,biological equivalents and/or antibodies. Additionally, the presentinvention relates to immunogenic compositions and their use inpreventing, treating and/or diagnosing meningococcal infection caused byN. meningitidis, and in particular meningococcal disease caused by N.meningitidis serogroup B, as well as methods for preparing saidcompositions. This invention relates to both recombinant forms and formsisolated from a natural source, as well as both lipidated andnon-lipidated forms.

BACKGROUND OF THE INVENTION

Meningococcal meningitis is a devastating disease that can kill childrenand young adults within hours despite the availability of antibiotics.Pizza et al., 2000, Science 287:1816-1820. Meningitis is Characterizedas an Inflammation of the meninges resulting in an intense headache,fever, loss of appetite, intolerance to light and sound, rigidity ofmuscles, especially in the neck, and in severe cases convulsions,vomiting and delirium leading to death. The symptoms of meningococcalmeningitis appear suddenly and culminate in meningococcal septicemiawith its characteristic hemorrhagic rash. A rapid diagnosis andimmediate treatment with large doses of antibiotics is critical if thereis to be any chance of survival. 2000. Bantam Medical Dictionary, ThirdEdition 302.

Meningococcal meningitis is caused by Neisseria meningitidis (themeningococcus), a Gram-negative, capsulated bacterium that has beenclassified into several pathogenic serogroups including A, B, C, D,W-135, X, Y, Z and 29E. Serogroup B strains of N. meningitidis are amajor cause of meningococcal disease throughout the world. For example,it is reported in the medical literature that serogroup B is responsiblefor about 50% of bacterial meningitis in infants and children residingin the United States and Europe. No vaccine currently exists to preventmeningococcal disease caused by N. meningitidis serogroup B.

Developing an immunogenic composition for the prevention of serogroup Bmeningococcal disease has been a challenge to researchers since the workof Goldschneider et al. over thirty years ago. Goldschneider et al.,1969, J. Exp. Med 129(6):1307-26; Goldschneider et al, 1969, J. Exp. Med129(6):1327-48; Gotschlich et al., 1969, J. Exp. Med. 129(6):1385-95;and Gotschlich et al., 1969, J. Exp. Med. 129(6):1367-84. Unlikeserogroup A disease, which virtually disappeared from North Americaafter World War II, Achtman, M., 1995, Trends in Microbiology3(5):186-92, disease caused by serogroup B and C organisms remainsendemic throughout much of the economically developed world. Theincidence of disease varies from <1/100,000 where endemic disease israre to 200/100,000 in high risk populations during epidemics.

Vaccines based on polysaccharide conjugates have been developed againstN. meningitidis serogroups A and C and appear to be effective inpreventing disease. Currently, an immunogenic composition made ofcapsular polysaccharide from serogroups A, C, Y, & W-135 is available.Ambrosch et al., 1983, Immunogenicity and side-effects of a newtetravalent. Bulletin of the World Health Organization 61(2):317-23.However, this immunogenic composition elicits a T-cell independentimmune response, is not effective in young children, and provides nocoverage for serogroup B strains, which cause upwards of 50% ofmeningococcal disease.

Others have also attempted to develop immunogenic compositions usingcapsular polysaccharides. Recently, immunogenic compositions forserogroup C disease prepared by conjugating the serogroup C capsularmaterial to proteins have been licensed for use in Europe. However, theserogroup B capsule may be unsuitable as a vaccine candidate because thecapsule polysaccharide is composed of polysialic acid which bears asimilarity to carbohydrate moieties on developing human neural tissues.This sugar moiety is recognized as a self-antigen and is thus poorlyimmunogenic in humans.

Outer membrane proteins (OMP's) have been developed as alternativevaccine antigens for serogroup B disease. Monoclonal antibody binding tothe two variable regions of PorA define the serosubtyping scheme formeningococci. PorA proteins thus serve as the serosubtyping antigens(Abdillahi et al., 1988, Microbial Pathogenesis 4(1):27-32) formeningococcal strains and are being actively investigated as componentsof a serogroup B immunogenic composition (Poolman, 1996, Adv. Exp. Med.Biol. 397:73-7), since they can elicit bactericidal antibodies(Saukkonen, 1987, Microbial Pathogenesis 3(4):261-7). Bactericidalantibodies are thought to be an indicator of protection and any newimmunogenic composition candidate should elicit these functionalantibodies.

Studies in humans as well as animals indicate that the serosubtypingantigen, PorA, elicits bactericidal antibodies. However, the immuneresponse to Por A is generally serosubtype specific. In particular,serosubtyping data indicate that an immunogenic composition made ofPorAs may require a PorA for each serosubtype to be covered by such animmunogenic composition, perhaps as many as six to nine. Therefore, 6-9PorAs will be needed to cover 70-80% of serogroup B strains. Thus, thevariable nature of this protein requires a multivalent vaccinecomposition to protect against a sufficient number of meningococcalserosubtype clinical isolates.

Developing an immunogenic composition for serogroup B meningococci hasbeen so difficult that recently several groups have sequenced thegenomes from strains representing both serogroups A and B to assist inidentifying new immunogenic composition candidates. Tettelin, 2000,Science, 287(5459):1809-15; Pizza et al., 2000, Science 287:1816-1820.Identifying new immunogenic composition candidates, even with theknowledge of the neisserial genome, is a challenging process for whichadequate mathematical algorithms do not currently exist. In fact, arecent report indicates that despite identifying hundreds of openreading frames (“ORFs”) containing theoretical membrane spanningdomains, problems with expression, purification, and inducing surfacereactive, and functionally active antibodies have led investigators toonly seven candidates for a serogroup B meningococcal immunogeniccomposition. See Id. One of these was previously known.

Accordingly, there remains a need for immunogenic compositions that (1)elicit bactericidal antibodies to multiple neisserial strains; (2) reactwith the surface of multiple strains; (3) confer passive protectionagainst a live challenge; and/or (4) prevent colonization.

SUMMARY OF THE INVENTION

To meet these and other needs, and in view of its purposes, the presentinvention provides Neisseria ORF2086 proteins (“2086 proteins”),including 2086 Subfamily A proteins and 2086 Subfamily B proteins. Eachof the 2086 proteins are proteins that can be isolated from nativeneisserial strains, including strains of Neisseria meningitidis(serogroups A, B, C, D, W-135, X, Y, Z and 29E), Neisseria gonorrhoeae,and Neisseria lactamica. The 2086 proteins may also be prepared usingrecombinant technology.

In particular, the present invention provides the 2086 proteins,immunogenic portions thereof, and/or biological equivalents thereof,antibodies that immunospecifically bind to any of the foregoing, andpolynucleotides comprising nucleic acid sequences that encode any of theforegoing. The present invention includes compositions, immunogeniccompositions and their use in preventing, treating and/or diagnosingmeningococcal infection, and in particular meningococcal disease causedby N. meningitidis, as well as methods for preparing said compositions.The 2086 proteins herein include recombinant forms and forms isolatedfrom a natural source, as well as both lipidated and non-lipidatedforms.

The present invention unexpectedly and advantageously providescompositions that (1) elicit bactericidal antibodies to multipleneisserial strains, such as strains of N. meningitidis, N. gonorrhoeae,and/or N. lactamica; (2) react with the surface of multiple strains; (3)confer passive protection against a live challenge; and/or (4) preventcolonization, as well as methods of using said compositions and methodsof preparing said compositions. Various embodiments of the invention aredescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an SDS-PAGE gel that depicts the two major proteins ofthe protein fractions obtained from the experiments for identifyingneisserial membrane protein extract that is capable of elicitingbactericidal antibodies against heterologous strains.

FIG. 1B depicts the results from the experiments from the identificationof the two major proteins by analysis of TMAE Flow Through components byprotease digestion and reverse Phase N-terminal sequencing.

FIG. 2 depicts the purification scheme and homogeneity as determined bySDS-PAGE of rLP2086.

FIG. 3 depicts the results from the experiments from the identificationof the two major proteins and one minor protein by analysis of TMAE FlowThrough components by LC-MS/MS and the corresponding SDS-PAGE.

FIG. 4 is an SDS-PAGE gel from the recombinant expression of 2086protein.

FIG. 5 is a schematic diagram of plasmid pPX7340, as described in theexamples herein.

FIG. 6 is a schematic diagram of plasmid pPX7328 as described in theexamples herein.

FIG. 7 is a schematic diagram of plasmid pPX7343 as described in theexamples herein.

FIG. 8 illustrates N-terminal regions of 2086 gene from various strains.The amino acid sequence shown for strain 8529 depicts the N-terminalregion of the 2086 ORF as shown in SEQ ID NO:212. The amino acidsequence shown for strain 2996 depicts the N-terminal region of the 2086ORF as shown in SEQ ID NO:110. The amino acid sequence shown for strain1573 depicts the N-terminal region of the 2086 ORF as shown in SEQ IDNO:248.

FIG. 9A is a flow chart showing the preliminary steps in theidentification of an immunogenic component in a nesserial strain.

FIG. 9B is a flow chart showing the final steps in the identification ofan immunogenic component in a nesserial strain.

FIG. 10A is a schematic diagram of the pBAD arabinose inducible promoterwhich drives the expression of the P4 signal/ORF2086 fusion protein toexpress a lipidated form of rP2086 as described in the examples herein.

FIG. 10B is a schematic diagram of the pET9a-T7 vector for recombinantexpression of nonlipidated form of ORF2086.

FIG. 11A is a photograph representing whole cell lysates of E. coli Bexpressing the rLP2086 protein.

FIG. 11B is a photograph representing whole cell lysates of E. coli Bexpressing the rP2086 protein.

FIG. 12 is a phylogenetic tree showing the organization of thesubfamilies and groups of ORF2086 proteins.

FIG. 13 is a graphical illustration of whole cell ELISA data for therLP2086 Subfamily A antisera.

FIG. 14 is a graphical illustration of whole cell ELISA data for therLP2086 Subfamily B antisera.

FIG. 15 is a graphical illustration of the results of the rLP2086 mixingstudy-WCE Titers.

FIG. 16 is a graphical illustration of the results of the rLP2086/rPorAmixing study—WCE Titers.

FIG. 17 is a Western Blot showing reactivity of rLP2086 mouse antiserato P2086 Subfamily B N. meningitidis whole cell lysates.

FIG. 18 is a Western Blot showing reactivity of rLP2086 mouse antiserato P2086 Subfamily A N. meningitidis and N. lactamica whole celllysates.

SEQUENCE SUMMARY SEQ ID NOS. For Studied Sequences:

SEQ ID NO:1 nucleic acid sequence encoding amino acid sequence formature 2086 protein from L3 6275 strain when combined with a nativeleader sequence.

SEQ ID NO:2 amino acid sequence for mature 2086 protein from L3 6275strain prepared using a native leader sequence.

SEQ ID NO:3 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from L3 6275 when combined with a P4 leadersequence.

SEQ ID NO:4 amino acid sequence for mature 2086 protein from L3 6275strain prepared using a P4 leader sequence.

SEQ ID NO:5 nucleic acid sequence encoding amino acid sequence formature 2086 protein from L3 6275 strain.

SEQ ID NO:6 amino acid sequence for mature 2086 protein from L3 6275strain.

SEQ ID NO:7 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC2369 strain when combined with a nativeleader sequence.

SEQ ID NO:8 amino acid sequence for mature 2086 protein from CDC2369strain prepared using a native leader sequence.

SEQ ID NO:9 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from CDC2369 when combined with a P4 leadersequence.

SEQ ID NO:10 amino acid sequence for mature 2086 protein from CDC2369strain prepared using a P4 leader sequence.

SEQ ID NO:11 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC2369 strain.

SEQ ID NO:12 amino acid sequence for mature 2086 protein from CDC2369strain.

SEQ ID NO:13 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1034 strain when combined with a nativeleader sequence.

SEQ ID NO:14 amino acid sequence for mature 2086 protein from CDC1034strain prepared using a native leader sequence.

SEQ ID NO:15 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from CDC1034 when combined with a P4 leadersequence.

SEQ ID NO:16 amino acid sequence for mature 2086 protein from CDC1034strain prepared using a P4 leader sequence.

SEQ ID NO:17 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1034 strain.

SEQ ID NO:18 amino acid sequence for mature 2086 protein from CDC1034strain.

SEQ ID NO:19 nucleic acid sequence encoding amino acid sequence formature 2086 protein from L4 891 strain when combined with a nativeleader sequence.

SEQ ID NO:20 amino acid sequence for mature 2086 protein from L4 891strain prepared using a native leader sequence.

SEQ ID NO:21 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from L4 891 when combined with a P4 leader sequence.

SEQ ID NO:22 amino acid sequence for mature 2086 protein from L4 891strain prepared using a P4 leader sequence.

SEQ ID NO:23 nucleic acid sequence encoding amino acid sequence formature 2086 protein from L4 891 strain.

SEQ ID NO:24 amino acid sequence for mature 2086 protein from L4 891strain.

SEQ ID NO:25 nucleic acid sequence encoding amino acid sequence formature 2086 protein from B16B6 strain when combined with a native leadersequence.

SEQ ID NO:26 amino acid sequence for mature 2086 protein from B16B6strain prepared using a native leader sequence.

SEQ ID NO:27 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from B16B6 when combined with a P4 leader sequence.

SEQ ID NO:28 amino acid sequence for mature 2086 protein from B16B6strain prepared using a P4 leader sequence.

SEQ ID NO:29 nucleic acid sequence encoding amino acid sequence formature 2086 protein from B16B6 strain.

SEQ ID NO:30 amino acid sequence for mature 2086 protein from B16B6strain.

SEQ ID NO:31 nucleic acid sequence encoding amino acid sequence formature 2086 protein from W135 (ATCC35559) strain when combined with anative leader sequence.

SEQ ID NO:32 amino acid sequence for mature 2086 protein from W135(ATCC35559) strain prepared using a native leader sequence.

SEQ ID NO:33 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from W135 (ATCC35559) when combined with a P4 leadersequence.

SEQ ID NO:34 amino acid sequence for mature 2086 protein from W135(ATCC35559) strain prepared using a P4 leader sequence.

SEQ ID NO:35 nucleic acid sequence encoding amino acid sequence formature 2086 protein from W135 (ATCC35559) strain.

SEQ ID NO:36 amino acid sequence for mature 2086 protein from W135(ATCC35559) strain.

SEQ ID NO:37 nucleic acid sequence encoding amino acid sequence formature 2086 protein from C11 strain when combined with a native leadersequence.

SEQ ID NO:38 amino acid sequence for mature 2086 protein from C11 strainprepared using a native leader sequence.

SEQ ID NO:39 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from C11 when combined with a P4 leader sequence.

SEQ ID NO:40 amino acid sequence for mature 2086 protein from C11 strainprepared using a P4 leader sequence.

SEQ ID NO:41 nucleic acid sequence encoding amino acid sequence formature 2086 protein from C11 strain.

SEQ ID NO:42 amino acid sequence for mature 2086 protein from C11strain.

SEQ ID NO:43 nucleic acid sequence encoding amino acid sequence formature 2086 protein from Y (ATCC35561) strain when combined with anative leader sequence.

SEQ ID NO:44 amino acid sequence for mature 2086 protein from Y(ATCC35561) strain prepared using a native leader sequence.

SEQ ID NO:45 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from Y (ATCC35561) when combined with a P4 leadersequence.

SEQ ID NO:46 amino acid sequence for mature 2086 protein from Y(ATCC35561) strain prepared using a P4 leader sequence.

SEQ ID NO:47 nucleic acid sequence encoding amino acid sequence formature 2086 protein from Y (ATCC35561) strain.

SEQ ID NO:48 amino acid sequence for mature 2086 protein from Y(ATCC35561) strain.

SEQ ID NO:49 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250732 strain when combined with a nativeleader sequence.

SEQ ID NO:50 amino acid sequence for mature 2086 protein from M98 250732strain prepared using a native leader sequence.

SEQ ID NO:51 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M98 250732 when combined with a P4 leadersequence.

SEQ ID NO:52 amino acid sequence for mature 2086 protein from M98 250732strain prepared using a P4 leader sequence.

SEQ ID NO:53 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250732 strain.

SEQ ID NO:54 amino acid sequence for mature 2086 protein from M98 250732strain.

SEQ ID NO:55 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250771 strain when combined with a nativeleader sequence.

SEQ ID NO:56 amino acid sequence for mature 2086 protein from M98 250771strain prepared using a native leader sequence.

SEQ ID NO:57 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M98 250771 when combined with a P4 leadersequence.

SEQ ID NO:58 amino acid sequence for mature 2086 protein from M98 250771strain prepared using a P4 leader sequence.

SEQ ID NO:59 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250771 strain.

SEQ ID NO:60 amino acid sequence for mature 2086 protein from M98 250771strain.

SEQ ID NO:61 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1135 strain when combined with a nativeleader sequence.

SEQ ID NO:62 amino acid sequence for mature 2086 protein from CDC1135strain prepared using a native leader sequence.

SEQ ID NO:63 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from CDC1135 when combined with a P4 leadersequence.

SEQ ID NO:64 amino acid sequence for mature 2086 protein from CDC1135strain prepared using a P4 leader sequence.

SEQ ID NO:65 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1135 strain.

SEQ ID NO:66 amino acid sequence for mature 2086 protein from CDC1135strain.

SEQ ID NO:67 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 252153 strain when combined with a nativeleader sequence.

SEQ ID NO:68 amino acid sequence for mature 2086 protein from M97 252153strain prepared using a native leader sequence.

SEQ ID NO:69 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M97 252153 when combined with a P4 leadersequence.

SEQ ID NO:70 amino acid sequence for mature 2086 protein from M97 252153strain prepared using a P4 leader sequence.

SEQ ID NO:71 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 252153 strain.

SEQ ID NO:72 amino acid sequence for mature 2086 protein from M97 252153strain.

SEQ ID NO:73 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1610 strain when combined with a nativeleader sequence.

SEQ ID NO:74 amino acid sequence for mature 2086 protein from CDC1610strain prepared using a native leader sequence.

SEQ ID NO:75 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from CDC1610 when combined with a P4 leadersequence.

SEQ ID NO:76 amino acid sequence for mature 2086 protein from CDC1610strain prepared using a P4 leader sequence.

SEQ ID NO:77 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1610 strain.

SEQ ID NO:78 amino acid sequence for mature 2086 protein from CDC1610strain.

SEQ ID NO:79 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1492 strain when combined with a nativeleader sequence.

SEQ ID NO:80 amino acid sequence for mature 2086 protein from CDC1492strain prepared using a native leader sequence.

SEQ ID NO:81 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from CDC1492 when combined with a P4 leadersequence.

SEQ ID NO:82 amino acid sequence for mature 2086 protein from CDC1492strain prepared using a P4 leader sequence.

SEQ ID NO:83 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1492 strain.

SEQ ID NO:84 amino acid sequence for mature 2086 protein from CDC1492strain.

SEQ ID NO:85 nucleic acid sequence encoding amino acid sequence formature 2086 protein from L8 M978 strain when combined with a nativeleader sequence.

SEQ ID NO:86 amino acid sequence for mature 2086 protein from L8 M978strain prepared using a native leader sequence.

SEQ ID NO:87 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from L8 M978 when combined with a P4 leadersequence.

SEQ ID NO:88 amino acid sequence for mature 2086 protein from L8 M978strain prepared using a P4 leader sequence.

SEQ ID NO:89 nucleic acid sequence encoding amino acid sequence formature 2086 protein from L8 M978 strain.

SEQ ID NO:90 amino acid sequence for mature 2086 protein from L8 M978strain.

SEQ ID NO:91 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 252988 strain when combined with a nativeleader sequence.

SEQ ID NO:92 amino acid sequence for mature 2086 protein from M97 252988strain prepared using a native leader sequence.

SEQ ID NO:93 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M97 252988 when combined with a P4 leadersequence.

SEQ ID NO:94 amino acid sequence for mature 2086 protein from M97 252988strain prepared using a P4 leader sequence.

SEQ ID NO:95 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 252988 strain.

SEQ ID NO:96 amino acid sequence for mature 2086 protein from M97 252988strain.

SEQ ID NO:97 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 252697 strain when combined with a nativeleader sequence.

SEQ ID NO:98 amino acid sequence for mature 2086 protein from M97 252697strain prepared using a native leader sequence.

SEQ ID NO:99 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M97 252697 when combined with a P4 leadersequence.

SEQ ID NO:100 amino acid sequence for mature 2086 protein from M97252697 strain prepared using a P4 leader sequence.

SEQ ID NO:101 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 252697 strain.

SEQ ID NO:102 amino acid sequence for mature 2086 protein from M97252697 strain.

SEQ ID NO:103 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 6557 strain when combined with a native leadersequence.

SEQ ID NO:104 amino acid sequence for mature 2086 protein from 6557strain prepared using a native leader sequence.

SEQ ID NO:105 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from 6557 when combined with a P4 leader sequence.

SEQ ID NO:106 amino acid sequence for mature 2086 protein from 6557strain prepared using a P4 leader sequence.

SEQ ID NO:107 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 6557 strain.

SEQ ID NO:108 amino acid sequence for mature 2086 protein from 6557strain.

SEQ ID NO:109 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 2996 strain when combined with a native leadersequence.

SEQ ID NO:110 amino acid sequence for mature 2086 protein from 2996strain prepared using a native leader sequence.

SEQ ID NO:111 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from 2996 when combined with a P4 leader sequence.

SEQ ID NO:112 amino acid sequence for mature 2086 protein from 2996strain prepared using a P4 leader sequence.

SEQ ID NO:113 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 2996 strain.

SEQ ID NO:114 amino acid sequence for mature 2086 protein from 2996strain.

SEQ ID NO:115 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 252976 strain when combined with a nativeleader sequence.

SEQ ID NO:116 amino acid sequence for mature 2086 protein from M97252976 strain prepared using a native leader sequence.

SEQ ID NO:117 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M97 252976 when combined with a P4 leadersequence.

SEQ ID NO:118 amino acid sequence for mature 2086 protein from M97252976 strain prepared using a P4 leader sequence.

SEQ ID NO:119 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 252976 strain.

SEQ ID NO:120 amino acid sequence for mature 2086 protein from M97252976 strain.

SEQ ID NO:121 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 251854 strain when combined with a nativeleader sequence.

SEQ ID NO:122 amino acid sequence for mature 2086 protein from M97251854 strain prepared using a native leader sequence.

SEQ ID NO:123 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M97 251854 when combined with a P4 leadersequence.

SEQ ID NO:124 amino acid sequence for mature 2086 protein from M97251854 strain prepared using a P4 leader sequence.

SEQ ID NO:125 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 251854 strain.

SEQ ID NO:126 amino acid sequence for mature 2086 protein from M97251854 strain.

SEQ ID NO:127 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1521 strain when combined with a nativeleader sequence.

SEQ ID NO:128 amino acid sequence for mature 2086 protein from CDC1521strain prepared using a native leader sequence.

SEQ ID NO:129 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from CDC1521 when combined with a P4 leadersequence.

SEQ ID NO:130 amino acid sequence for mature 2086 protein from CDC1521strain prepared using a P4 leader sequence.

SEQ ID NO:131 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1521 strain.

SEQ ID NO:132 amino acid sequence for mature 2086 protein from CDC1521strain.

SEQ ID NO:133 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250622 strain when combined with a nativeleader sequence.

SEQ ID NO:134 amino acid sequence for mature 2086 protein from M98250622 strain prepared using a native leader sequence.

SEQ ID NO:135 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M98 250622 when combined with a P4 leadersequence.

SEQ ID NO:136 amino acid sequence for mature 2086 protein from M98250622 strain prepared using a P4 leader sequence.

SEQ ID NO:137 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250622 strain.

SEQ ID NO:138 amino acid sequence for mature 2086 protein from M98250622 strain.

SEQ ID NO:139 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 870446 strain when combined with a nativeleader sequence.

SEQ ID NO:140 amino acid sequence for mature 2086 protein from 870446strain prepared using a native leader sequence.

SEQ ID NO:141 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from 870446 when combined with a P4 leader sequence.

SEQ ID NO:142 amino acid sequence for mature 2086 protein from 870446strain prepared using a P4 leader sequence.

SEQ ID NO:143 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 870446 strain.

SEQ ID NO:144 amino acid sequence for mature 2086 protein from 870446strain.

SEQ ID NO:145 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 253248 strain when combined with a nativeleader sequence.

SEQ ID NO:146 amino acid sequence for mature 2086 protein from M97253248 strain prepared using a native leader sequence.

SEQ ID NO:147 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M97 253248 when combined with a P4 leadersequence.

SEQ ID NO:148 amino acid sequence for mature 2086 protein from M97253248 strain prepared using a P4 leader sequence.

SEQ ID NO:149 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 253248 strain.

SEQ ID NO:150 amino acid sequence for mature 2086 protein from M97253248 strain.

SEQ ID NO:151 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250809 strain when combined with a nativeleader sequence.

SEQ ID NO:152 amino acid sequence for mature 2086 protein from M98250809 strain prepared using a native leader sequence.

SEQ ID NO:153 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M98 250809 when combined with a P4 leadersequence.

SEQ ID NO:154 amino acid sequence for mature 2086 protein from M98250809 strain prepared using a P4 leader sequence.

SEQ ID NO:155 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250809 strain.

SEQ ID NO:156 amino acid sequence for mature 2086 protein from M98250809 strain.

SEQ ID NO:157 nucleic acid sequence encoding amino acid sequence formature 2086 protein from L5 M981 strain when combined with a nativeleader sequence.

SEQ ID NO:158 amino acid sequence for mature 2086 protein from L5 M981strain prepared using a native leader sequence.

SEQ ID NO:159 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from L5 M981 when combined with a P4 leadersequence.

SEQ ID NO:160 amino acid sequence for mature 2086 protein from L5 M981strain prepared using a P4 leader sequence.

SEQ ID NO:161 nucleic acid sequence encoding amino acid sequence formature 2086 protein from L5 M981 strain.

SEQ ID NO:162 amino acid sequence for mature 2086 protein from L5 M981strain.

SEQ ID NO:163 nucleic acid sequence encoding amino acid sequence formature 2086 protein from NMB strain when combined with a native leadersequence.

SEQ ID NO:164 amino acid sequence for mature 2086 protein from NMBstrain prepared using a native leader sequence.

SEQ ID NO:165 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from NMB when combined with a P4 leader sequence.

SEQ ID NO:166 amino acid sequence for mature 2086 protein from NMBstrain prepared using a P4 leader sequence.

SEQ ID NO:167 nucleic acid sequence encoding amino acid sequence formature 2086 protein from NMB strain.

SEQ ID NO:168 amino acid sequence for mature 2086 protein from NMBstrain.

SEQ ID NO:169 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250572 strain when combined with a nativeleader sequence.

SEQ ID NO:170 amino acid sequence for mature 2086 protein from M98250572 strain prepared using a native leader sequence.

SEQ ID NO:171 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M98 250572 when combined with a P4 leadersequence.

SEQ ID NO:172 amino acid sequence for mature 2086 protein from M98250572 strain prepared using a P4 leader sequence.

SEQ ID NO:173 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250572 strain.

SEQ ID NO:174 amino acid sequence for mature 2086 protein from M98250572 strain.

SEQ ID NO:175 nucleic acid sequence encoding amino acid sequence formature 2086 protein from A4 Sanford; M97 251836 PART; M97 251957; M97251985; M97 252060; M97 251870; M97 251994; M98 250024; M97 251905; M97251876; M97 251898; or M97 251830 strain when combined with a nativeleader sequence.

SEQ ID NO:176 amino acid sequence for mature 2086 protein from A4Sanford; M97 251836 PART; M97 251957; M97 251985; M97 252060; M97251870; M97 251994; M98 250024; M97 251905; M97 251876; M97 251898; orM97 251830 strain prepared using a native leader sequence.

SEQ ID NO:177 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from A4 Sanford; M97 251836 PART; M97 251957; M97251985; M97 252060; M97 251870; M97 251994; M98 250024; M97 251905; M97251876; M97 251898; or M97 251830 when combined with a P4 leadersequence.

SEQ ID NO:178 amino acid sequence for mature 2086 protein from A4Sanford; M97 251836 PART; M97 251957; M97 251985; M97 252060; M97251870; M97 251994; M98 250024; M97 251905; M97 251876; M97 251898; orM97 251830 strain prepared using a P4 leader sequence.

SEQ ID NO:179 nucleic acid sequence encoding amino acid sequence formature 2086 protein from A4 Sanford; M97 251836 part; M97 251957; M97251985; M97 252060; M97 251870; M97 251994; M98 250024; M97 251905; M97251876; M97 251898; or M97 251830 strain.

SEQ ID NO:180 amino acid sequence for mature 2086 protein from A4Sanford; M97 251836 PART; M97 251957; M97 251985; M97 252060; M97251870; M97 251994; M98 250024; M97 251905; M97 251876; M97 251898; orM97 251830 strain.

SEQ ID NO:181 nucleic acid sequence encoding partial amino acid sequencefor mature 2086 protein from CDC937 strain when combined with a nativeleader sequence.

SEQ ID NO:182 amino acid sequence for mature 2086 protein from CDC937strain prepared using a native leader sequence.

SEQ ID NO:183 nucleic acid sequence for encoding partial amino acidsequence for mature 2086 protein from CDC937 when combined with a P4leader sequence.

SEQ ID NO:184 amino acid sequence for mature 2086 protein from CDC937strain prepared using a P4 leader sequence.

SEQ ID NO:185 nucleic acid sequence encoding partial amino acid sequencefor mature 2086 protein from CDC937 strain.

SEQ ID NO:186 amino acid sequence for mature 2086 protein from CDC937strain.

SEQ ID NO:187 nucleic acid sequence encoding partial amino acid sequencefor mature 2086 protein from M97 252097 strain when combined with anative leader sequence.

SEQ ID NO:188 amino acid sequence for mature 2086 protein from M97252097 strain prepared using a native leader sequence.

SEQ ID NO:189 nucleic acid sequence for encoding partial amino acidsequence for mature 2086 protein from M97 252097 when combined with a P4leader sequence.

SEQ ID NO:190 amino acid sequence for mature 2086 protein from M97252097 strain prepared using a P4 leader sequence.

SEQ ID NO:191 nucleic acid sequence encoding partial amino acid sequencefor mature 2086 protein from M97 252097 strain.

SEQ ID NO:192 amino acid sequence for mature 2086 protein from M97252097 strain.

SEQ ID NO:193 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 870227 strain when combined with a nativeleader sequence.

SEQ ID NO:194 amino acid sequence for mature 2086 protein from 870227strain prepared using a native leader sequence.

SEQ ID NO:195 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from 870227 when combined with a P4 leader sequence.

SEQ ID NO:196 amino acid sequence for mature 2086 protein from 870227strain prepared using a P4 leader sequence.

SEQ ID NO:197 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 870227 strain.

SEQ ID NO:198 amino acid sequence for mature 2086 protein from 870227strain.

SEQ ID NO:199 nucleic acid sequence encoding amino acid sequence formature 2086 protein from H355 strain when combined with a native leadersequence.

SEQ ID NO:200 amino acid sequence for mature 2086 protein from H355strain prepared using a native leader sequence.

SEQ ID NO:201 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from H355 when combined with a P4 leader sequence.

SEQ ID NO:202 amino acid sequence for mature 2086 protein from H355strain prepared using a P4 leader sequence.

SEQ ID NO:203 nucleic acid sequence encoding amino acid sequence formature 2086 protein from H355 strain.

SEQ ID NO:204 amino acid sequence for mature 2086 protein from H355strain.

SEQ ID NO:205 nucleic acid sequence encoding amino acid sequence formature 2086 protein from H44_(—)76 strain when combined with a nativeleader sequence.

SEQ ID NO:206 amino acid sequence for mature 2086 protein from H44_(—)76strain prepared using a native leader sequence.

SEQ ID NO:207 amino acid sequence for mature 2086 protein from H44_(—)76strain prepared using a P4 leader sequence.

SEQ ID NO:208 nucleic acid sequence encoding amino acid sequence formature 2086 protein from H44_(—)76 strain.

SEQ ID NO:209 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from H44_(—)76 when combined with a P4 leadersequence.

SEQ ID NO:210 amino acid sequence for mature 2086 protein from H44_(—)76strain.

SEQ ID NO:211 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 8529 strain when combined with a native leadersequence.

SEQ ID NO:212 amino acid sequence for mature 2086 protein from 8529strain prepared using a native leader sequence.

SEQ ID NO:213 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from 8529 when combined with a P4 leader sequence.

SEQ ID NO:214 amino acid sequence for mature 2086 protein from 8529strain prepared using a P4 leader sequence.

SEQ ID NO:215 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 8529 strain.

SEQ ID NO:216 amino acid sequence for mature 2086 protein from 8529strain.

SEQ ID NO:217 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 6940 strain when combined with a native leadersequence.

SEQ ID NO:218 amino acid sequence for mature 2086 protein from 6940strain prepared using a native leader sequence.

SEQ ID NO:219 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from 6940 when combined with a P4 leader sequence.

SEQ ID NO:220 amino acid sequence for mature 2086 protein from 6940strain prepared using a P4 leader sequence.

SEQ ID NO:221 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 6940 strain.

SEQ ID NO:222 amino acid sequence for mature 2086 protein from 6940strain.

SEQ ID NO:223 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M982 strain when combined with a native leadersequence.

SEQ ID NO:224 amino acid sequence for mature 2086 protein from M982strain prepared using a native leader sequence.

SEQ ID NO:225 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M982 when combined with a P4 leader sequence.

SEQ ID NO:226 amino acid sequence for mature 2086 protein from M982strain prepared using a P4 leader sequence.

SEQ ID NO:227 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M982 strain.

SEQ ID NO:228 amino acid sequence for mature 2086 protein from M982strain.

SEQ ID NO:229 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 880049 strain when combined with a nativeleader sequence.

SEQ ID NO:230 amino acid sequence for mature 2086 protein from 880049strain prepared using a native leader sequence.

SEQ ID NO:231 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from 880049 when combined with a P4 leader sequence.

SEQ ID NO:232 amino acid sequence for mature 2086 protein from 880049strain prepared using a P4 leader sequence.

SEQ ID NO:233 nucleic acid sequence encoding amino acid sequence formature 2086 protein from 880049 strain.

SEQ ID NO:234 amino acid sequence for mature 2086 protein from 880049strain.

SEQ ID NO:235 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 253524, M97 251885, and M97 251926 strainswhen combined with a native leader sequence.

SEQ ID NO:236 amino acid sequence for mature 2086 protein from M97253524, M97 251885, and M97 251926 strains prepared using a nativeleader sequence.

SEQ ID NO:237 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M97 253524, M97 251885, and M97 251926 strainswhen combined with a P4 leader sequence.

SEQ ID NO:238 amino acid sequence for mature 2086 protein from M97253524, M97 251885, and M97 251926 strains prepared using a P4 leadersequence.

SEQ ID NO:239 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M97 253524, M97 251885, and M97 251926 strains.

SEQ ID NO:240 amino acid sequence for mature 2086 protein from M97253524, M97 251885, and M97 251926 strains.

SEQ ID NO:241 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250670 strain when combined with a nativeleader sequence.

SEQ ID NO:242 amino acid sequence for mature 2086 protein from M98250670 strain prepared using a native leader sequence.

SEQ ID NO:243 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from M98 250670 when combined with a P4 leadersequence.

SEQ ID NO:244 amino acid sequence for mature 2086 protein from M98250670 strain prepared using a P4 leader sequence.

SEQ ID NO:245 nucleic acid sequence encoding amino acid sequence formature 2086 protein from M98 250670 strain.

SEQ ID NO:246 amino acid sequence for mature 2086 protein from M98250670 strain.

SEQ ID NO:247 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1573 strain when combined with a nativeleader sequence.

SEQ ID NO:248 amino acid sequence for mature 2086 protein from CDC1573strain prepared using a native leader sequence.

SEQ ID NO:249 nucleic acid sequence for encoding amino acid sequence formature 2086 protein from CDC1573 when combined with a P4 leadersequence.

SEQ ID NO:250 amino acid sequence for mature 2086 protein from CDC1573strain prepared using a P4 leader sequence.

SEQ ID NO:251 nucleic acid sequence encoding amino acid sequence formature 2086 protein from CDC1573 strain.

SEQ ID NO:252 amino acid sequence for mature 2086 protein from CDC1573strain.

SEQ ID NO:253 partial nucleic acid sequence encoding amino acid sequencefor 2086 protein from a strain of Neisseria lactamica.

SEQ ID NOS:254 to 259 amino acid sequences associated with proteins of2086 family of proteins.

SEQ ID NOS:260 to 278 amino acid sequences associated with proteins of2086 Subfamily A.

SEQ ID NOS:279 to 299 amino acid sequences associated with proteins of2086 Subfamily B.

SEQ ID NO:300 is the amino acid consensus sequence corresponding to the2086 protein family (“2086 proteins”) according to an embodiment of thepresent invention.

SEQ ID NO:301 is the amino acid consensus sequence corresponding to the2086 protein Subfamily A according to an embodiment of the presentinvention.

SEQ ID NO:302 is the amino acid consensus sequence corresponding to the2086 protein Subfamily B according to an embodiment of the presentinvention.

SEQ ID NO:303 nucleic acid sequence for a reverse primer with BamHIrestriction site (Compound No. 4623).

SEQ ID NO:304 nucleic acid sequence for a forward primer with NdeIrestriction site (Compound No. 4624).

SEQ ID NO:305 nucleic acid sequence for a forward primer (Compound No.4625).

SEQ ID NO:306 nucleic acid sequence for a forward primer (Compound No.5005).

SEQ ID NO:307 nucleic acid sequence for a reverse primer (Compound No.5007).

SEQ ID NO:308 nucleic acid sequence for a reverse primer with BglIIrestriction site (Compound No. 5135).

SEQ ID NO:309 nucleic acid sequence for a forward primer with BamHIrestriction site (Compound No. 5658).

SEQ ID NO:310 nucleic acid sequence for a reverse primer with SphIrestriction site (Compound No. 5660).

SEQ ID NO:311 nucleic acid sequence for a forward primer with BamHIrestriction site (Compound No. 6385).

SEQ ID NO:312 nucleic acid sequence for a forward primer with BglII andNdeI restriction sites (Compound No. 6406).

SEQ ID NO:313 nucleic acid sequence for a forward primer (Compound No.6470).

SEQ ID NO:314 nucleic acid sequence for a reverse primer (Compound No.6472).

SEQ ID NO:315 nucleic acid sequence for a forward primer with BamHIrestriction site (Compound 6473).

SEQ ID NO:316 nucleic acid sequence for a forward primer with BglII andNdeI restriction sites (Compound No. 6474).

SEQ ID NO:317 nucleic acid sequence for a forward primer (Compound No.6495).

SEQ ID NO:318 nucleic acid sequence for a reverse primer (Compound No.6496).

SEQ ID NO:319 nucleic acid sequence for a reverse primer with SphIrestriction site (Compound No. 6543).

SEQ ID NO:320 nucleic acid sequence for a reverse primer with BglIIrestriction site (Compound No. 6605).

SEQ ID NO:321 nucleic acid sequence for a forward primer with BglII andNdeI restriction sites (Compound No. 6721).

SEQ ID NO:322 nucleic acid sequence for the P4 leader sequence.

SEQ ID NO:323 nucleic acid sequence for native 2086 leader variant 1.

SEQ ID NO:324 nucleic acid sequence for native 2086 leader variant 2.

SEQ ID NO:325 nucleic acid sequence for native 2086 leader variant 3.

SEQ ID NO:326 nucleic acid sequence for native 2086 leader variant 4.

SEQ ID NO:327 is the amino acid sequence of P4431.

SEQ ID NO:328 is the amino acid sequence of P5163.

SEQ ID NO:329 is an amino acid sequence according to an embodiment ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

A new class of antigens with cross-functional bactericidal activityagainst Neisseria meningitidis serogroup B would obviate the need for amulti-valent PorA approach to immunization against infection. Such anantigen has been unexpectedly identified and is described and claimedherein. The presence of one such antigen was first observed in a complexmixture of soluble outer membrane proteins (sOMPs) from a meningococcalstrain. The bactericidal activity of this antigen was followed through aseries of fractionation and protein purification steps until the proteinmixture of interest contained just a few proteins. The major proteins inthis mixture were identified by N-terminal amino acid sequencing andpeptide mapping. The protein of interest exhibiting bactericidalactivity was identified as ORF2086 protein, a lipidated protein (alsomore specifically referred to as LP2086). “ORF2086 protein” refers to aprotein encoded by open reading frame 2086 (ORF2086) of Neisseriaspecies.

As described herein, new immunogenic composition candidates based onNeisseria species ORF2086 protein (also referred to as “2086 protein” or“ORF2086” protein, used interchangably herein, or P2086 for thenon-lipated proteins and LP2086 for the lipidated version of theproteins) isolated from N. meningitidis were identified by combiningcell fractionation, differential detergent extraction, proteinpurification, with the preparation of antisera, and a bactericidalactivity assay utilizing multiple strains. As an alternative topotential immunogenic compositions and diagnostics disclosed in thereferences cited above, this invention relates to compositions andmethods of treating and/or preventing meningococcal infection throughthe use of proteins, immunogenic portions thereof and biologicalequivalents thereof, as well as genes encoding said polypeptides,portions and equivalents, and antibodies that immunospecifically bind tosame.

According to an embodiment of the present invention, immunogenic agentsbased on 2086 protein, including isolated polypeptides, immunogenicportions thereof and/or biological equivalents thereof were unexpectedlyidentified as immunogenic candidates based on the ability of said agentsto exhibit cross-reactivity or non-strain specificity. In particular,candidates were identified that unexpectedly demonstrate the ability to(1) elicit bactericidal antibodies to multiple neisserial and/orgonococcal strains; (2) react with the surface of multiple strains; (3)confer passive protection against a live challenge; and/or (4) preventcolonization. Accordingly, the present invention provides immunogeniccompositions comprising said immunogenic agents, including isolatedpolypeptides, immunogenic portions thereof, and/or biologicalequivalents thereof, as well as methods for using same against infectionby N. meningitidis. (See Example 1 herein for the methodology used inthe identification of the 2086 protein.)

As used herein, the term “non-strain specific” refers to thecharacteristic of an antigen to elicit an immune response effectiveagainst more than one strain of N. meningitidis (e.g., heterologousmeningococcal strains). The term “cross-reactive” as it is used hereinis used interchangeably with the term “non-strain specific”. The term“immunogenic non-strain specific N. meningitidis antigen,” as usedherein, describes an antigen that can be isolated from N. meningitidis,although it can also be isolated from another bacterium (e.g., otherneisserial strains, such as gonococcal strains, for example), orprepared using recombinant technology.

The 2086 proteins of the present invention include lipidated andnon-lipidated proteins. Further, the present invention also contemplatesthe use of the immature proteins or preproteins that correspond to eachprotein as intermediate compounds/compositions.

The present invention also provides antibodies that immunospecificallybind to the foregoing immunogenic agents, according to implementationsof the invention. Further, the present invention relates to isolatedpolynucleotides comprising nucleic acid sequences encoding any of theforegoing. Additionally, the present invention provides compositionsand/or immunogenic compositions and their use in preventing, treatingand/or diagnosing meningococcal meningitis, in particular serogroup Bmeningococcal disease, as well as methods for preparing saidcompositions.

The compositions of the present invention have been shown to be highlyimmunogenic and capable of eliciting the production of bactericidalantibodies. These antibodies are cross-reactive to serogroup, serotypeand serosubtype heterologous meningococcal strains. Accordingly, thepresent compositions overcome the deficiencies of previous N.meningitidis vaccine attempts by exhibiting the ability to elicitbactericidal antibodies to heterologous neisserial strains. Thus, amongother advantages, the present invention provides immunogeniccompositions that can be compounded with fewer components to elicitprotection comparable to previously used agents. The compositions orimmunogenic agents therein (e.g., polypeptides, immunogenic portions orfragments, and biological equivalents, etc., without limitation) can beused alone or in combination with other antigens or agents to elicitimmunological protection from meningococcal infection and disease, aswell as to elicit immunological protection from infection and/or diseasecaused by other pathogens. This simplifies the design of an immunogeniccomposition for use against meningococcal infection by reducing thenumber of antigens required for protection against multiple strains. Infact, purified 2086 protein will dramatically and unexpectedly reducethe number of proteins required to provide adequate immunogenic coverageof the strains responsible for meningococcal disease. The 2086 proteincan be recombinantly expressed in E. coli as a lipoprotein, which is thewild type form of the protein, at levels much higher than in the nativemeningococci.

Because antibodies directed against the 2086 protein from a singlestrain were found to kill unrelated (i.e., heterologous) strains, anattempt was made to characterize a large number of heterologous strainsfor the presence of a “2086 homolog”, and to determine the level ofsequence conservation. While about 70% of the strains tested by PCR had2086 homologs that could be amplified using the primers that amplifiedthe original 2086 gene from strain 8529, the remaining approximately 30%were negative by this test. These remaining approximately 30% were foundto contain a 2086 homolog that has only about 60% amino acid sequencehomology to the original 8529 derived 2086 gene. Other primers wereidentified that could amplify a 2086 homolog from these approximately30% of strains. The N. meningitidis strains tested have been designatedas belonging to Subfamily A or Subfamily B depending on which primer setcan amplify a 2086 homolog. The details of these experiments areoutlined in Example 5 below.

The Presence of a 2086 Protein in Numerous Serosubtypes.

To determine the level of sequence conservation of the 2086 gene betweenN. meningitidis strains, several representatives from Subfamilies A andB were cloned as full length genes and submitted for DNA sequenceanalysis. Using primers as disclosed herein, see, for example, Table IV,twenty four serogroup B meningococcal strains were identified, whichexpress different serosubtype antigens and also express a sharedprotein, P2086. Examples of these sequences are provided herein and areshown as mature DNA sequences (i.e., all lipoprotein signal sequenceshave been cleaved at the cysteine residue). See, for example, the aminoacid sequences of even numbered SEQ ID NOS: 2-252, without limitation.

Although the 2086 protein is not present in large amounts in wild typestrains, it is a target for bactericidal antibodies. These antibodies,unlike those produced in response to the PorAs, are capable of killingstrains expressing heterologous serosubtypes.

Antibodies to the 2086 protein also passively protect infant rats fromchallenge with meningococci. (see Table VII) Recombinant expression of2086 protein enables the use of 2086 protein as an immunogeniccomposition for the prevention of meningococcal disease. All of therecent meningococcal immunogenic composition candidates in clinicaltrials have been complex mixtures or outer membrane protein preparationscontaining many different proteins. The PorA protein, that providesserosubtype specificity, will require the inclusion of 6 to 9 variantsin an immunogenic composition to provide about 70-80% coverage ofdisease related serosubtypes. In contrast, it is clearly demonstratedherein that antisera to a single 2086 protein alone is able to killrepresentatives of six serosubtypes responsible for about 65% of thedisease isolates in western Europe and the United States. Therefore,purified 2086 protein has the potential to reduce the number of proteinsrequired to provide adequate immunogenic composition coverage of theserosubtypes responsible for meningococcal disease.

Proteins, Immunogenic Portions and Biological Equivalents

The 2086 proteins provided by the present invention are isolatedproteins. The term “isolated” means altered by the hand of man from thenatural state. If an “isolated” composition or substance occurs innature, it has been changed or removed from its original environment, orboth. For example, a polypeptide or a polynucleotide naturally presentin a living animal is not “isolated,” but the same polypeptide orpolynucleotide separated from the coexisting materials of its naturalstate is “isolated”, as the term is employed herein. Accordingly, asused herein, the term “isolated protein” encompasses proteins isolatedfrom a natural source and proteins prepared using recombinanttechnology, as well as such proteins when combined with other antigensand/or additives, such as pharmaceutically acceptable carriers, buffers,adjuvants, etc., for example.

A 2086 protein, immunogenic portion thereof and/or a biologicalequivalent thereof, according an embodiment of the invention, comprisesany of the following amino acid sequences:

(SEQ ID NO: 254) ADIGxGLADA,wherein x is any amino acid;

(SEQ ID NO: 255) IGxGLADALT,wherein x is any amino acid;

(SEQ ID NO: 256) SLNTGKLKND; (SEQ ID NO: 257 SLNTGKLKNDKxSRFDF,wherein x is any amino acid);

(SEQ ID NO: 258) SGEFQxYKQ,wherein x is any amino acid; or

(SEQ ID NO: 259) IEHLKxPE,wherein x is any amino acid.

A 2086 Subfamily A protein, immunogenic portion thereof and/orbiological equivalent thereof comprises any of the following amino acidsequences, in accordance with an embodiment of the present invention:

(SEQ ID NO: 260) GGGVAADIGx,wherein x is any amino acid;

(SEQ ID NO: 261) SGEFQIYKQ; (SEQ ID NO: 262) HSAVVALQIE;(SEQ ID NO: 263) EKINNPDKID; (SEQ ID NO: 264) SLINQRSFLV;(SEQ ID NO: 265) SGLGGEHTAF; (SEQ ID NO: 266) GEHTAFNQLP;(SEQ ID NO: 267) SFLVSGLGGEH; (SEQ ID NO: 268)EKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLP; (SEQ ID NO: 269) GKAEYHGKAF;(SEQ ID NO: 270) YHGKAFSSDD; (SEQ ID NO: 271) GKAEYHGKAFSSDD;(SEQ ID NO: 272) IEHLKTPEQN; (SEQ ID NO: 273) KTPEQNVELA;(SEQ ID NO: 274) IEHLKTPEQNVELA; (SEQ ID NO: 275) AELKADEKSH;(SEQ ID NO: 276) AVILGDTRYG; (SEQ ID NO: 277) AELKADEKSHAVILGDTRYG;  or(SEQ ID NO: 278) EEKGTYHLAL.

A 2086 Subfamily B protein, immunogenic portion thereof and/orbiological equivalent thereof comprises any of the following amino acidsequences, in accordance with an embodiment embodiment of the presentinvention:

(SEQ ID NO: 279) LITLESGEFQ; (SEQ ID NO: 280) SALTALQTEQ;(SEQ ID NO: 281) FQVYKQSHSA; (SEQ ID NO: 282)LITLESGEFQVYKQSHSALTALQTEQ; (SEQ ID NO: 283) IGDIAGEHTS;(SEQ ID NO: 284) EHTSFDKLPK; (SEQ ID NO: 285) IGDIAGEHTSFDKLPK;(SEQ ID NO: 286) ATYRGTAFGS; (SEQ ID NO: 287) DDAGGKLTYT;(SEQ ID NO: 288) IDFAAKQGHG; (SEQ ID NO: 289) KIEHLKSPEL;(SEQ ID NO: 290) ATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNV;(SEQ ID NO: 291) HAVISGSVLY; (SEQ ID NO: 292) KGSYSLGIFG;(SEQ ID NO: 293) VLYNQDEKGS; (SEQ ID NO: 294) HAVISGSVLYNQDEKGSYSLGIFG;(SEQ ID NO: 295) AQEVAGSAEV; (SEQ ID NO: 296) IHHIGLAAKQ;(SEQ ID NO: 297) VETANGIHHI; (SEQ ID NO: 298) AQEVAGSAEVETANGIHHIGLAAKQ;or (SEQ ID NO: 299) VAGSAEVETANGIHHIGLAAKQ.

The 2086 protein comprises the following consensus sequence and/orimmunogenic portions thereof in accordance with an embodiment of thepresent invention.

2086 Protein Consensus Sequence (SEQ ID NO: 300):CSSG-----GGGVxADIGxGLADALTxPxDxKDKxLxSLTLxxSxxxNxxLxLxAQGAEKTxxxGD---SLNTGKLKNDKxSRFDFxxxIxVDGxxITLxSGEFQxYKQxHSAxxALQxExxxxxxxxxxxxxxRxFxxxxxxGEHTxFxxLPxx-xAxYxGxAFxSDDxxGxLxYxIDFxxKQGxGxIEHLKxPExNVxLAxxxxKxDEKxHAVIxGxxxYxxxEKGxYxLxxxGxxAQExAGxAxVxx xxxxHxIxxAxKQ

In the foregoing consensus sequence, the “x” represents any amino acid,the region from amino acid position 5 to amino acid position 9 is any of0 to 5 amino acids, the region from amino acid position 67 to amino acidposition 69 is any of 0 to 3 amino acids, and amino acid position 156 isany of 0 to 1 amino acid. The region from amino acid position 5 to aminoacid position 9 preferably comprises 0, 4 or 5 amino acids. The regionfrom amino acid position 67 to amino acid position 69 preferablycomprises 0 or 3 amino acids. It should be particularly noted that thisconsensus sequence illustrates the high variability of the 2086proteins. By way of theory, without intending to be bound thereto, it isbelieved that this high variability provides the advantageous andunexpected cross-reactivity.

According to an implementation of the present invention, the 2086proteins are characterized as being immunogenic, nonpathogenic andnon-strain specific. Moreover, according to a further implementation ofthe present invention, these proteins unexpectedly exhibitimmunogenicity while being about 2% to about 40% nonconserved.

As used herein, the term “nonconserved” refers to the number of aminoacids that may undergo insertions, substitution and/or deletions as apercentage of the total number of amino acids in a protein. For example,if a protein is 40% nonconserved and has, for example, 263 amino acids,then there are 105 amino acid positions in the protein at which aminoacids may undergo substitution. Likewise, if a protein is 10%nonconserved and has, for example, about 280 amino acids, then there are28 amino acid positions at which amino acids may undergo substitution.The 2086 proteins may also undergo deletion of amino acid residueswithout compromising the immunogenicity of the proteins.

Further, the 2086 proteins may be divided into subfamilies based uponhomology at various regions. For example, without intending to belimited thereto, the consensus sequences for two such subfamilies,Subfamily A and Subfamily B, are provided below:

2086 Subfamily A sequence (SEQ ID 301)CSSG----GGGVAADIGxGLADALTxPxDxKDKxLxSLTLxxSxxxNxxLxLxAQGAEKTxxxGD---SLNTGKLKNDKxSRFDFxxxIxVDGQxITLxSGEFQIYKQxHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPxGKAEYHGKAFSSDDxxGxLxYxIDFxxKQGxGxIEHLKTPEQNVELAxAELKADEKSHAVILGDTRYGxEEKGTYHLALxGDRAQEIAGxATVKIxE KVHEIxIAxKQ

The reference “x” is any amino acid.

The region from amino acid position 5 to amino acid position 8 is any of0 to 4 amino acids.

The region from amino acid position 66 to amino acid position 68 is anyof 0 to 3 amino acids.

The region from amino acid position 5 to amino acid position 8preferably comprises 0 or 4 amino acids. The region from amino acidposition 66 to amino acid position 68 preferably comprises 0 or 3 aminoacids.

2086 Subfamily B (SEQ ID 302)CSSGGGG-----VxADIGxGLADALTAPLDHKDKxLxSLTLxxSxxxNxxLxLxAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGxLITLESGEFQVYKQSHSALTALQTEQxQDxExSxKMVAKRxFxIGDIAGEHTSFDKLPKxxxATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVxLAxxYIKPDEKxHAVISGSVLYNQDEKGSYSLGIFGxxAQEVAGSAEVETANG IHHIGLAAKQ

The reference “x” is any amino acid.

The region from amino acid position 8 to amino acid position 12 is anyof 0 to 5 amino acids.

The region from amino acid position 8 to amino acid position 12preferably comprises 0 or 5 amino acids.

According to implementations of the present invention, the 2086 proteinsubfamilies may be further subdivided into clusters. For example,according to an implementation of the present invention, the followingclusters are provided: even numbered SEQ ID NOS:2-12; even numbered SEQID NOS:14-24; even numbered SEQ ID NOS:26-42; even numbered SEQ IDNOS:50-60; even numbered SEQ ID NOS:62-108; even numbered SEQ IDNOS:110-138; even numbered SEQ ID NOS:140-156; even numbered SEQ IDNOS:158-174; and even numbered SEQ ID NOS: 224-252.

A polypeptide sequence of the invention may be identical to thereference sequence of even numbered SEQ ID NOS: 2-252, that is, 100%identical, or it may include a number of amino acid alterations ascompared to the reference sequence such that the % identity is less than100%. Such alterations include at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion. The alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference amino acid sequenceor in one or more contiguous groups within the reference amino acidsequence.

Thus, the invention also provides proteins having sequence identity tothe amino acid sequences contained in the Sequence Listing (i.e., evennumbered SEQ ID NOS: 2-252). Depending on the particular sequence, thedegree of sequence identity is preferably greater than 60% (e.g., 60%,70%, 80%, 90%, 95%, 97%, 99%, 99.9% or more). These homologous proteinsinclude mutants and allelic variants.

In preferred embodiments of the invention, the 2086 proteins or other2086 polypeptides (e.g., immunological portions and biologicalequivalents) generate bactericidal antibodies to homologous and at leastone heterologous strain of meningococci. Specifically, the antibodies tothe 2086 polypeptides passively protect infant rats from challenge, suchas intranasal, with meningococci. In further preferred embodiments, the2086 polypeptides exhibit such protection for infants rats forhomologous strains and at least one heterologous strain. The polypeptidemay be selected from the Sequence Summary above, as set forth in theeven numbered SEQ ID NOS: 2-252, or the polypeptide may be anyimmunological fragment or biological equivalent of the listedpolypeptides. Preferably, the polypeptide is selected from any of theeven numbered SEQ ID NOS: 2-252 in the Sequence Summary above.

This invention also relates to allelic or other variants of the 2086polypeptides, which are biological equivalents. Suitable biologicalequivalents will exhibit the ability to (1) elicit bactericidalantibodies to homologous strains and at least one heterologousneisserial strain and/or gonococcal strain; (2) react with the surfaceof homologous strains and at least one heterologous neisserial and/orgonococcal strain; (3) confer passive protection against a livechallenge; and/or (4) prevent colonization.

Suitable biological equivalents have at least about 60%, preferably atleast about 70%, more preferably at least about 75%, even morepreferably about 80%, even more preferably about 85%, even morepreferably about 90%, even more preferably 95% or even more preferably98%, or even more preferably 99% similarity to one of the 2086polypeptides specified herein (i.e., the even numbered SEQ ID NOS:2-252), provided the equivalent is capable of eliciting substantiallythe same immunogenic properties as one of the 2086 proteins of thisinvention.

Alternatively, the biological equivalents have substantially the sameimmunogenic properties of one of the 2086 protein in the even numberedSEQ ID NOS: 2-252. According to embodiments of the present invention,the biological equivalents have the same immunogenic properties as theeven numbered SEQ ID NOS 2-252.

The biological equivalents are obtained by generating variants andmodifications to the proteins of this invention. These variants andmodifications to the proteins are obtained by altering the amino acidsequences by insertion, deletion or substitution of one or more aminoacids. The amino acid sequence is modified, for example by substitutionin order to create a polypeptide having substantially the same orimproved qualities. A preferred means of introducing alterationscomprises making predetermined mutations of the nucleic acid sequence ofthe polypeptide by site-directed mutagenesis.

Modifications and changes can be made in the structure of a polypeptideof the present invention and still obtain a molecule having N.meningitidis immunogencity. For example, without limitation, certainamino acids can be substituted for other amino acids, includingnonconserved and conserved substitution, in a sequence withoutappreciable loss of immunogenicity. Because it is the interactivecapacity and nature of a polypeptide that defines that polypeptide'sbiological functional activity, a number of amino acid sequencesubstitutions can be made in a polypeptide sequence (or, of course, itsunderlying DNA coding sequence) and nevertheless obtain a polypeptidewith like properties. The present invention contemplates any changes tothe structure of the polypeptides herein, as well as the nucleic acidsequences encoding said polypeptides, wherein the polypeptide retainsimmunogenicity. A person of ordinary skill in the art would be readilyable to modify the disclosed polypeptides and polynucleotidesaccordingly, based upon the guidance provided herein.

For example, certain variable regions have been identified wheresubstitution or deletion is permissible The 2086 consensus sequence, aspreviously discussed, shows conserved and nonconserved regions of the2086 family of proteins according to an implementation of the presentinvention.

In making such changes, any techniques known to persons of skill in theart may be utilized. For example, without intending to be limitedthereto, the hydropathic index of amino acids can be considered. Theimportance of the hydropathic amino acid index in conferring interactivebiologic function on a polypeptide is generally understood in the art.Kyte et al. 1982. J. Mol. Bio. 157:105-132.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. U.S. Pat. No. 4,554,101, incorporated hereinby reference, states that the greatest local average hydrophilicity of apolypeptide, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity, i.e. with a biologicalproperty of the polypeptide.

Biological equivalents of a polypeptide can also be prepared usingsite-specific mutagenesis. Site-specific mutagenesis is a techniqueuseful in the preparation of second generation polypeptides, orbiologically functional equivalent polypeptides or peptides, derivedfrom the sequences thereof, through specific mutagenesis of theunderlying DNA. Such changes can be desirable where amino acidsubstitutions are desirable. The technique further provides a readyability to prepare and test sequence variants, for example,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art. As will be appreciated, the technique typically employs a phagevector which can exist in both a single stranded and double strandedform. Typically, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector which includeswithin its sequence a DNA sequence which encodes all or a portion of theN. meningitidis polypeptide sequence selected. An oligonucleotide primerbearing the desired mutated sequence is prepared (e.g., synthetically).This primer is then annealed to the single-stranded vector, and extendedby the use of enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cellssuch as E. coli cells and clones are selected which include recombinantvectors bearing the mutation. Commercially available kits come with allthe reagents necessary, except the oligonucleotide primers.

2086 polypeptides include any protein or polypeptide comprisingsubstantial sequence similarity and/or biological equivalence to a 2086protein having an amino acid sequence from one of the even numbered SEQID NOS 2-252. In addition, a 2086 polypeptide of the invention is notlimited to a particular source. Thus, the invention provides for thegeneral detection and isolation of the polypeptides from a variety ofsources. Also, the 2086 polypeptides can be prepared recombinantly, asis well within the skill in the art, based upon the guidance providedherein, or in any other synthetic manner, as known in the art.

It is contemplated in the present invention, that a 2086 polypeptide mayadvantageously be cleaved into fragments for use in further structuralor functional analysis, or in the generation of reagents such as2086-related polypeptides and 2086-specific antibodies. This can beaccomplished by treating purified or unpurified N. meningitidispolypeptides with a peptidase such as endoproteinase glu-C (Boehringer,Indianapolis, Ind.). Treatment with CNBr is another method by whichpeptide fragments may be produced from natural N. meningitidis 2086polypeptides. Recombinant techniques also can be used to producespecific fragments of a 2086 protein.

“Variant” as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical (i.e.,biologically equivalent). A variant and reference polypeptide may differin amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al 1984), BLASTP, BLASTN, andFASTA (Altschul, S. F., et al., 1990). The BLASTX program is publiclyavailable from NCBI and other sources (BLAST Manual, Altschul, S., etal., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., 1990). Thewell known Smith Waterman algorithm may also be used to determineidentity.

By way of example, without intending to be limited thereto, an aminoacid sequence of the present invention may be identical to the referencesequences, even numbered SEQ ID NOS: 2-252; that is be 100% identical,or it may include a number of amino acid alterations as compared to thereference sequence such that the % identity is less than 100%. Suchalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NOS:2-252 by thenumerical percent of the respective percent identity (divided by 100)and then subtracting that product from said total number of amino acidsin any of SEQ ID NOS:2-252, or:

n _(a) =x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NOS:2-252, and y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein anynon-integer product of x.sub.a and y is rounded down to the nearestinteger prior to subtracting it from x_(a).

In preferred embodiments, the polypeptide above is selected from theproteins set forth in the even numbered SEQ ID NOS 2-252, such as matureprocessed form of a 2086 protein. The 2086 proteins or equivalents, etc.may be lipidated or non-lipidated.

ORF 2086 is expressible in E. coli with the native ORF 2086 signalsequence. However, it is desirable to find means to improve theexpression of proteins. According to an embodiment of the presentinvention, a leader sequence produces a lipidated form of the protein.For example, the following describes the use of the signal sequence ofthe nontypable Haemophilus influenzae P4 protein to enhance expression.

The processing of bacterial lipoproteins begins with the synthesis of aprecursor or prolipoprotein containing a signal sequence, which in turncontains a consensus lipoprotein processing/modification site. Thisprolipoprotein initially passes through the common Sec system on theinner membrane of Gram negative bacteria or on the membrane in Grampositive bacteria. Once placed in the membrane by the Sec system, theprolipoprotein is cleaved by signal peptidase II at the consensus siteand the exposed N-terminal cysteine residue is glycerated and acylated.Hayashi et al. 1990. Lipoproteins in bacteria. J. Bioenerg. Biomembr.June; 22(3):451-71; Oudega et al. 1993. Escherichia coli SecB, SecA, andSecY proteins are required for expression and membrane insertion of thebacteriocin release protein, a small lipoprotein. J. Bacteriol. March;175(5):1543-7; Sankaran et al. 1995. Modification of bacteriallipoproteins. Methods Enzymol. 250:683-97.

In Gram negative bacteria, transport of the lipidated protein to theouter membrane is mediated by a unique ABC transporter system withmembrane specificity depending on a sorting signal at position 2 of thelipoprotein. Yakushi et al. 2000. A new ABC transporter mediating thedetachment of lipid modified proteins from membranes. Nat Cell Biol.April; 2(4):212-8.

Fusion with bacterial lipoproteins and their signal sequences has beenused to display recombinant proteins on the surface of bacteria. U.S.Pat. Nos. 5,583,038 and 6,130,085. Exchanging lipoprotein signalsequences can increase the production of the lipoprotein. De et al.2000. Purification and characterization of Streptococcus pneumoniaepalmitoylated pneumococcal surface adhesin A expressed in Escherichiacoli. Vaccine. March 6; 18(17): 1811-21.

Bacterial lipidation of proteins is known to increase or modify theimmunological response to proteins. Erdile et al. 1993. Role of attachedlipid in immunogenicity of Borrelia burgdorferi OspA. Infect. Immun.January; 61(1):81-90; Snapper et al. 1995. Bacterial lipoproteins maysubstitute for cytokines in the humoral immune response to Tcell-independent type II antigens. J. Immunol. December 15;155(12):5582-9. However, bacterial lipoprotein expression can becomplicated by the stringency of the processing. Pollitt et al. 1986.Effect of amino acid substitutions at the signal peptide cleavage siteof the Escherichia coli major outer membrane lipoprotein. J. Biol. Chem.February 5; 261(4):1835-7; Lunn et al. 1987. Effects of prolipoproteinsignal peptide mutations on secretion of hybrid prolipo-beta-lactamasein Escherichia coli. J. Biol. Chem. June 15; 262(17):8318-24; Klein etal. 1988. Distinctive properties of signal sequences from bacteriallipoproteins. Protein Eng. April; 2(1):15-20. Bacterial lipoproteinexpression is also complicated by other problems such as toxicity andlow expression levels. Gomez et al. 1994. Nucleotide The Bacillussubtilis lipoprotein LplA causes cell lysis when expressed inEscherichia coli. Microbiology. August; 140 (Pt 8):1839-45; Hansson etal. 1995. Expression of truncated and full-length forms of the Lymedisease Borrelia outer surface protein A in Escherichia coli. ProteinExpr. Purif. February; 6(1):15-24; Yakushi et al. 1997. Lethality of thecovalent linkage between mislocalized major outer membrane lipoproteinand the peptidoglycan of Escherichia coli. J. Bacteriol. May;179(9):2857-62.

The nontypable Haemophilus influenzae bacterium expresses a lipoproteindesignated P4 (also known as protein “e”). The recombinant form of theP4 protein is highly expressed in E. coli using the native P4 signalsequence. U.S. Pat. No. 5,955,580. When the native P4 signal sequence issubstituted for the native ORF 2086 signal sequence in an expressionvector in E. coli, the level of expression of ORF2086 is increased.

This concept of using the heterologous P4 signal sequence to increaseexpression is extendible to other bacterial lipoproteins. In particular,analysis of bacterial genomes leads to the identification of many ORFsas being of possible interest. Attempting to express each ORF with itsnative signal sequence in a heterologous host cell, such as E. coli,gives rise to a variety of problems inherent in using a variety ofsignal sequences, including stability, compatibility and so forth. Tominimize these problems, the P4 signal sequence is used to express eachORF of interest. As described above, the P4 signal sequence improves theexpression of the heterologous 2086 ORF. An expression vector isconstructed by deleting the native signal sequence of the ORF ofinterest, and ligating the P4 signal sequence to the ORF. A suitablehost cell is then transformed, transfected or infected with theexpression vector, and expression of the ORF is increased in comparisonto expression using the native signal sequence of the ORF.

The non-lipidated form is produced by a protein lacking the originalleader sequence or a by a leader sequence which is replaced with aportion of sequence that does not specify a site for fatty acidacylation in a host cell.

The various forms of the 2086 proteins of this invention are referred toherein as “2086” protein, unless otherwise specifically noted. Also“2086 polypeptide” refers to the 2086 proteins as well as immunogenicportions or biological equivalents thereof as noted above, unlessotherwise noted.

The full length isolated and purified N. meningitidis 2086 protein hasan apparent molecular weight of about 28 to 35 kDa as measured on a 10%to 20% gradient SDS polyacrylamide gel (SDS-PAGE). More specifically,this protein has a molecular weight of about 26,000 to 30,000 daltons asmeasured by mass spectrometry.

Preferably, the 2086 polypeptides and nucleic acids encoding suchpolypeptides are used for preventing or ameliorating infection caused byN. meningitidis and/or other species.

Antibodies

The proteins of the invention, including the amino acid sequences of SEQID NOS: 2-252, their fragments, and analogs thereof, or cells expressingthem, are also used as immunogens to produce antibodies immunospecificfor the polypeptides of the invention. The invention includes antibodiesto immunospecific polypeptides and the use of such antibodies to detectthe presence of N. meningitidis, provide passive protection or measurethe quantity or concentration of the polypeptides in a cell, a cell ortissue extract, or a biological fluid.

The antibodies of the invention include polyclonal antibodies,monoclonal antibodies, chimeric antibodies, and anti-idiotypicantibodies. Polyclonal antibodies are heterogeneous populations ofantibody molecules derived from the sera of animals immunized with anantigen. Monoclonal antibodies are a substantially homogeneouspopulation of antibodies to specific antigens. Monoclonal antibodies maybe obtained by methods known to those skilled in the art, e.g., Kohlerand Milstein, 1975, Nature 256:495-497 and U.S. Pat. No. 4,376,110. Suchantibodies may be of any immunoglobulin class including IgG, IgM, IgE,IgA, GILD and any subclass thereof.

Chimeric antibodies are molecules, different portions of which arederived from different animal species, such as those having variableregion derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Chimeric antibodies and methods fortheir production are known in the art (Cabilly et al., 1984, Proc. Natl.Acad. Sci. USA 81:3273-3277; Morrison et al., 1984, Proc. Natl. Acad.Sci. USA 81:6851-6855; Boulianne et al., 1984, Nature 312:643-646;Cabilly et al., European Patent Application 125023 (published Nov. 14,1984); Taniguchi et al., European Patent Application 171496 (publishedFeb. 19, 1985); Morrison et al., European Patent Application 173494(published Mar. 5, 1986); Neuberger et al., PCT Application WO 86/01533(published Mar. 13, 1986); Kudo et al., European Patent Application184187 (published Jun. 11, 1986); Morrison et al., European PatentApplication 173494 (published Mar. 5, 1986); Sahagan et al., 1986, J.Immunol. 137:1066-1074; Robinson et al., PCT/US86/02269 (published May7, 1987); Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Sunet al., 1987, Proc. Nati. Acad. Sci. USA 84:214-218; Better et al.,1988, Science 240:1041-1043). These references are hereby incorporatedby reference in their entirety.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An anti-Id antibody is prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the monoclonal antibody with the monoclonal antibody to which ananti-Id is being prepared. The immunized animal will recognize andrespond to the idiotypic determinants of the immunizing antibody byproducing an antibody to these isotypic determinants (the anti-Idantibody).

Accordingly, monoclonal antibodies generated against the polypeptides ofthe present invention may be used to induce anti-Id antibodies insuitable animals. Spleen cells from such immunized mice can be used toproduce anti-Id hybridomas secreting anti-Id monoclonal antibodies.Further, the anti-Id antibodies can be coupled to a carrier such askeyhole limpet hemocyanin (KLH) and used to immunize additional BALB/cmice. Sera from these mice will contain anti-anti-Id antibodies thathave the binding properties of the final mAb specific for an R-PTPaseepitope. The anti-Id antibodies thus have their idiotypic epitopes, or“idiotopes” structurally similar to the epitope being evaluated, such asStreptococcus pyogenes polypeptides.

The term “antibody” is also meant to include both intact molecules aswell as fragments such as Fab which are capable of binding antigen. Fabfragments lack the Fc fragment of intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding thanan intact antibody (Wahl et al., 1983, J. Nucl. Med. 24:316-325). Itwill be appreciated that Fab and other fragments of the antibodiesuseful in the present invention may be used for the detection andquantitation of N. meningitidis polypeptides according to the methodsfor intact antibody molecules.

The antibodies of this invention, such as anti-iodiotypic (“anti-Id”)antibodies, can be employed in a method for the treatment or preventionof Neisseria infection in mammalian hosts, which comprisesadministration of an immunologically effective amount of antibody,specific for a polypeptide as described above. The anti-Id antibody mayalso be used as an “immunogen” to induce an immune response in yetanother animal, producing a so-called anti-anti-Id antibody. Theanti-anti-Id may be epitopically identical to the original mAb whichinduced the anti-Id. Thus, by using antibodies to the idiotypicdeterminants of a mAb, it is possible to identify other clonesexpressing antibodies of identical specificity.

The antibodies are used in a variety of ways, e.g., for confirmationthat a protein is expressed, or to confirm where a protein is expressed.Labeled antibody (e.g., fluorescent labeling for FACS) can be incubatedwith intact bacteria and the presence of the label on the bacterialsurface confirms the location of the protein, for instance.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogs, or cells to an animal using routine protocols. For preparingmonoclonal antibodies, any technique which provides antibodies producedby continuous cell line cultures are used.

Polynucleotides

As with the proteins of the present invention, a polynucleotide of thepresent invention may comprise a nucleic acid sequence that is identicalto any of the reference sequences of odd numbered SEQ ID NOS:1-253, thatis be 100% identical, or it may include up to a number of nucleotidealterations as compared to the reference sequence. Such alterations areselected from the group consisting of at least one nucleotide deletion,substitution, including transition and transversion, or insertion, andwherein said alterations may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among the nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The number of nucleotide alterations is determinedby multiplying the total number of nucleotides in any of odd numberedSEQ ID NOS:1-253 by the numerical percent of the respective percentidentity (divided by 100) and subtracting that product from said totalnumber of nucleotides in said sequence.

By way of example, without intending to be limited thereto, an isolatedN. meningitidis polynucleotide comprising a polynucleotide sequence thathas at least 70% identity to any nucleic acid sequence of SEQ IDNOS:1-253; a degenerate variant thereof or a fragment thereof, whereinthe polynucleotide sequence may include up to n_(n) nucleic acidalterations over the entire polynucleotide region of the nucleic acidsequence of SEQ ID NOS:1-253, wherein n_(n) is the maximum number ofalterations and is calculated by the formula:

n _(n) =x _(n)−(x _(n) ·y),

in which x_(n) is the total number of nucleic acids of any of SEQ IDNOS:1-253 and y has a value of 0.70, wherein any non-integer product ofx_(n) and y is rounded down to the nearest integer prior to subtractingsuch product from x_(n). Of course, y may also have a value of 0.80 for80%, 0.85 for 85%, 0.90 for 90% 0.95 for 95%, etc. Alterations of apolynucleotide sequence encoding the polypeptides comprising amino acidsequences of any of SEQ ID NOS:2-252 may create nonsense, missense orframeshift mutations in this coding sequence and thereby alter thepolypeptide encoded by the polynucleotide following such alterations.

Certain embodiments of the present invention relate to polynucleotides(herein referred to as the “2086 polynucleotides” or “ORF2086polynucleotides”) which encode the 2086 proteins and antibodies madeagainst the 2086 proteins. In preferred embodiments, an isolatedpolynucleotide of the present invention is a polynucleotide comprising anucleotide sequence having at least about 95% identity to a nucleotidesequence chosen from one of the odd numbered SEQ ID NO: 1 and SEQ IDNOS: 253, a degenerate variant thereof, or a fragment thereof. Asdefined herein, a “degenerate variant” is defined as a polynucleotidethat differs from the nucleotide sequence shown in the odd numbered SEQID NOS:1 and SEQ ID NOS:253 (and fragments thereof) due to degeneracy ofthe genetic code, but still encodes the same 2086 protein (i.e., theeven numbered SEQ ID NOS: 2-252) as that encoded by the nucleotidesequence shown in the odd numbered SEQ ID NOS: 1-253.

In other embodiments, the polynucleotide is a complement to a nucleotidesequence chosen from one of the odd numbered SEQ ID NOS: 1-253, adegenerate variant thereof, or a fragment thereof. In yet otherembodiments, the polynucleotide is selected from the group consisting ofDNA, chromosomal DNA, cDNA and RNA and may further comprisesheterologous nucleotides. In another embodiment, an isolatedpolynucleotide hybridizes to a nucleotide sequence chosen from one ofSEQ ID NOS: 1-253, a complement thereof, a degenerate variant thereof,or a fragment thereof, under high stringency hybridization conditions.In yet other embodiments, the polynucleotide hybridizes underintermediate stringency hybridization conditions.

It will be appreciated that the 2086 polynucleotides may be obtainedfrom natural, synthetic or semi-synthetic sources; furthermore, thenucleotide sequence may be a naturally occurring sequence, or it may berelated by mutation, including single or multiple base substitutions,deletions, insertions and inversions, to such a naturally occurringsequence, provided always that the nucleic acid molecule comprising sucha sequence is capable of being expressed as 2086 immunogenic polypeptideas described above. The nucleic acid molecule may be RNA, DNA, singlestranded or double stranded, linear or covalently closed circular form.The nucleotide sequence may have expression control sequences positionedadjacent to it, such control sequences usually being derived from aheterologous source. Generally, recombinant expression of the nucleicacid sequence of this invention will use a stop codon sequence, such asTAA, at the end of the nucleic acid sequence.

The invention also includes polynucleotides capable of hybridizing underreduced stringency conditions, more preferably stringent conditions, andmost preferably highly stringent conditions, to polynucleotidesdescribed herein. Examples of stringency conditions are shown in theStringency Conditions Table below: highly stringent conditions are thosethat are at least as stringent as, for example, conditions A-F;stringent conditions are at least as stringent as, for example,conditions G-L; and reduced stringency conditions are at least asstringent as, for example, conditions M-R.

TABLE I STRINGENCY CONDITIONS Polynucle- Hybrid Hybridization WashStringency otide Length Temperature and Temperature Condition Hybrid(bp)^(I) Buffer^(H) and Buffer^(H) A DNA:DNA >50 65EC; 1xSSC -or- 65EC;0.3xSSC 42EC; 1xSSC, 50% formamide B DNA:DNA <50 T_(B); 1xSSC T_(B);1xSSC C DNA:RNA >50 67EC; 1xSSC -or- 67EC; 0.3xSSC 45EC; 1xSSC, 50%formamide D DNA:RNA <50 T_(D); 1xSSC T_(D); 1xSSC E RNA:RNA >50 70EC;1xSSC -or- 70EC; 0.3xSSC 50EC; 1xSSC, 50% formamide F RNA:RNA <50 T_(F);1xSSC T_(f); 1xSSC G DNA:DNA >50 65EC; 4xSSC -or- 65EC; 1xSSC 42EC;4xSSC, 50% formamide H DNA:DNA <50 T_(H); 4xSSC T_(H); 4xSSC IDNA:RNA >50 67EC; 4xSSC -or- 67EC; 1xSSC 45EC; 4xSSC, 50% formamide JDNA:RNA <50 T_(J); 4xSSC T_(J); 4xSSC K RNA:RNA >50 70EC; 4xSSC -or-67EC; 1xSSC 50EC; 4xSSC, 50% formamide L RNA:RNA <50 T_(L); 2xSSC T_(L);2xSSC M DNA:DNA >50 50EC; 4xSSC -or- 50EC; 2xSSC 40EC; 6xSSC, 50%formamide N DNA:DNA <50 T_(N); 6xSSC T_(N); 6xSSC O DNA:RNA >50 55EC;4xSSC -or- 55EC; 2xSSC 42EC; 6xSSC, 50% formamide P DNA:RNA <50 T_(P);6xSSC T_(P); 6xSSC Q RNA:RNA >50 60EC; 4xSSC -or- 60EC; 2xSSC 45EC;6xSSC, 50% formamide R RNA:RNA <50 T_(R); 4xSSC T_(R); 4xSSC bp^(I): Thehybrid length is that anticipated for the hybridized region(s) of thehybridizing polynucleotides. When hybridizing a polynucleotide to atarget polynucleotide of unknown sequence, the hybrid length is assumedto be that of the hybridizing polynucleotide. When polynucleotides ofknown sequence are hybridized, the hybrid length can be determined byaligning the sequences of the polynucleotides and identifying the regionor regions of optimal sequence complementarities. buffer^(H): SSPE(1xSSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can besubstituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) inthe hybridization and wash buffers; washes are performed for 15 minutesafter hybridization is complete. T_(B) through T_(R): The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10EC less than the melting temperature (T_(m)) of thehybrid, where T_(m) is determined according to the following equations.For hybrids less than 18 base pairs in length, T_(m)(EC) = 2(# of A + Tbases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairsin length, T_(m)(EC) = 81.5 + 16.6(log₁₀[Na⁺]) + 0.41(% G + C) −(600/N), where N is the number of bases in the hybrid, and [Na⁺] is theconcentration of sodium ions in the hybridization buffer ([Na⁺] for1xSSC = 0.165M).

Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference.

The invention also provides polynucleotides that are fully complementaryto these polynucleotides and also provides antisense sequences. Theantisense sequences of the invention, also referred to as antisenseoligonucleotides, include both internally generated and externallyadministered sequences that block expression of polynucleotides encodingthe polypeptides of the invention. The antisense sequences of theinvention comprise, for example, about 15-20 base pairs. The antisensesequences can be designed, for example, to inhibit transcription bypreventing promoter binding to an upstream nontranslated sequence or bypreventing translation of a transcript encoding a polypeptide of theinvention by preventing the ribosome from binding.

The polynucleotides of the invention are prepared in many ways (e.g., bychemical synthesis, from DNA libraries, from the organism itself) andcan take various forms (e.g., single-stranded, double-stranded, vectors,probes, primers). The term “polynucleotide” includes DNA and RNA, andalso their analogs, such as those containing modified backbones.

According to further implementations of the present invention, thepolynucleotides of the present invention comprise a DNA library, such asa cDNA library.

Fusion Proteins

The present invention also relates to fusion proteins. A “fusionprotein” refers to a protein encoded by two, often unrelated, fusedgenes or fragments thereof. For example, fusion proteins comprisingvarious portions of constant region of immunoglobulin molecules togetherwith another immunogenic protein or part thereof. In many cases,employing an immunoglobulin Fc region as a part of a fusion protein isadvantageous for use in therapy and diagnosis resulting in, for example,improved pharmacokinetic properties (see, e.g., EP 0 232 262 A1). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified. The 2086 polynucleotides of the invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself, or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence which facilitates purification of a 2086 polypeptideor fused polypeptide can be encoded (see Gentz et al., 1989,incorporated herein by reference in its entirety). Thus, contemplated inan implementation of the present invention is the preparation ofpolynucleotides encoding fusion polypeptides permitting His-tagpurification of expression products. The polynucleotide may also containnon-coding 5′ and 3′ sequences, such as transcribed, non-translatedsequences, splicing and polyadenylation signals. Such a fusedpolypeptide can be produced by a host cell transformed/transfected orinfected or infected with a recombinant DNA cloning vehicle as describedbelow and it can be subsequently isolated from the host cell to providethe fused polypeptide substantially free of other host cell proteins.

Immunogenic Compositions

One aspect of the present invention provides immunogenic compositionswhich comprise at least one 2086 proteins or a nucleic acid encodingsaid proteins. The foregoing have the ability to (1) elicit bactericidalantibodies to multiple strains; (2) react with the surface of multiplestrains; (3) confer passive protection against a live challenge; and/or(4) prevent colonization.

The formulation of such immunogenic compositions is well known topersons skilled in this field Immunogenic compositions of the inventionpreferably include a pharmaceutically acceptable carrier. Suitablepharmaceutically acceptable carriers and/or diluents include any and allconventional solvents, dispersion media, fillers, solid carriers,aqueous solutions, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like. Suitablepharmaceutically acceptable carriers include, for example, one or moreof water, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibody.The preparation and use of pharmaceutically acceptable carriers is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the immunogeniccompositions of the present invention is contemplated.

Such immunogenic compositions can be administered parenterally, e.g., byinjection, either subcutaneously or intramuscularly, as well as orallyor intranasally. Methods for intramuscular immunization are described byWolff et al. and by Sedegah et al. Other modes of administration employoral formulations, pulmonary formulations, suppositories, andtransdermal applications, for example, without limitation. Oralformulations, for example, include such normally employed excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,and the like, without limitation.

The immunogenic compositions of the invention can include one or moreadjuvants, including, but not limited to aluminum hydroxide; aluminumphosphate; STIMULON™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham,Mass.); MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton,Mont.), 529 (an amino alkyl glucosamine phosphate compound, Corixa,Hamilton, Mont.), IL-12 (Genetics Institute, Cambridge, Mass.); GM-CSF(Immunex Corp., Seattle, Wash.);N-acetyl-muramyl-L-theronyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy-ethylamine)(CGP 19835A, referred to as MTP-PE); and cholera toxin. Others which maybe used are non-toxic derivatives of cholera toxin, including its Asubunit, and/or conjugates or genetically engineered fusions of the N.meningitidis polypeptide with cholera toxin or its B subunit (“CTB”),procholeragenoid, fungal polysaccharides, including schizophyllan,muramyl dipeptide, muramyl dipeptide (“MDP”) derivatives, phorbolesters, the heat labile toxin of E. coli, block polymers or saponins.

In certain preferred embodiments, the proteins of this invention areused in an immunogenic composition for oral administration whichincludes a mucosal adjuvant and used for the treatment or prevention ofN. meningitidis infection in a human host. The mucosal adjuvant can be acholera toxin; however, preferably, mucosal adjuvants other than choleratoxin which may be used in accordance with the present invention includenon-toxic derivatives of a cholera holotoxin, wherein the A subunit ismutagenized, chemically modified cholera toxin, or related proteinsproduced by modification of the cholera toxin amino acid sequence. For aspecific cholera toxin which may be particularly useful in preparingimmunogenic compositions of this invention, see the mutant choleraholotoxin E29H, as disclosed in Published International Application WO00/18434, which is hereby incorporated herein by reference in itsentirety. These may be added to, or conjugated with, the polypeptides ofthis invention. The same techniques can be applied to other moleculeswith mucosal adjuvant or delivery properties such as Escherichia coliheat labile toxin (LT). Other compounds with mucosal adjuvant ordelivery activity may be used such as bile; polycations such asDEAE-dextran and polyornithine; detergents such as sodium dodecylbenzene sulphate; lipid-conjugated materials; antibiotics such asstreptomycin; vitamin A; and other compounds that alter the structuralor functional integrity of mucosal surfaces. Other mucosally activecompounds include derivatives of microbial structures such as MDP;acridine and cimetidine. STIMULON™ QS-21, MPL, and IL-12, as describedabove, may also be used.

The immunogenic compositions of this invention may be delivered in theform of ISCOMS (immune stimulating complexes), ISCOMS containing CTB,liposomes or encapsulated in compounds such as acrylates orpoly(DL-lactide-co-glycoside) to form microspheres of a size suited toadsorption. The proteins of this invention may also be incorporated intooily emulsions.

Multiple Antigens

The immunogenic agents, including proteins, polynucleotides andequivalents of the present invention may be administered as the soleactive immunogen in a immunogenic composition, or alternatively, thecomposition may include other active immunogens, including otherNeisseria sp immunogenic polypeptides, or immunologically-activeproteins of one or more other microbial pathogens (e.g. virus, prion,bacterium, or fungus, without limitation) or capsular polysaccharide.The compositions may comprise one or more desired proteins, fragments orpharmaceutical compounds as desired for a chosen indication. In the samemanner, the compositions of this invention which employ one or morenucleic acids in the immunogenic composition may also include nucleicacids which encode the same diverse group of proteins, as noted above.

Any multi-antigen or multi-valent immunogenic composition iscontemplated by the present invention. For example, the compositions ofthe present invention may a comprise combinations of two or more 2086proteins, a combination of 2086 protein with one or more Por A proteins,a combination of 2086 protein with meningococcus serogroup A, C, Y andW135 polysaccharides and/or polysaccharide conjugates, a combination of2086 protein with meningococcus and pneumococcus combinations, or acombination of any of the foregoing in a form suitable for mucosaldelivery. Persons of skill in the art would be readily able to formulatesuch multi-antigen or multi-valent immunologic compositions.

The present invention also contemplates multi-immunization regimenswherein any composition useful against a pathogen may be combinedtherein or therewith the compositions of the present invention. Forexample, without limitation, a patient may be administered theimmunogenic composition of the present invention and anotherimmununological composition for immunizing against S. Pneumoniae, aspart of a multi-immunization regimen. Persons of skill in the art wouldbe readily able to select immunogenic compositions for use inconjunction with the immunogenic compositions of the present inventionfor the purposes of developing and implementing multi-immunizationregimens.

Specific embodiments of this invention relate to the use of one or morepolypeptides of this invention, or nucleic acids encoding such, in acomposition or as part of a treatment regimen for the prevention oramelioration of S. pneumonaie infection. One can combine the 2086polypeptides or 2086 polynucleotides with any immunogenic compositionfor use against S. pneumonaie infection. One can also combine the 2086polypeptides or 2086 polynucleotides with any other protein orpolysaccharide-based meningococcal vaccine.

The 2086 polypeptides, fragments and equivalents can be used as part ofa conjugate immunogenic composition; wherein one or more proteins orpolypeptides are conjugated to a carrier in order to generate acomposition that has immunogenic properties against several serotypesand/or against several diseases. Alternatively, one of the 2086polypeptides can be used as a carrier protein for other immunogenicpolypeptides.

The present invention also relates to a method of inducing immuneresponses in a mammal comprising the step of providing to said mammal animmunogenic composition of this invention. The immunogenic compositionis a composition which is antigenic in the treated animal or human suchthat the immunologically effective amount of the polypeptide(s)contained in such composition brings about the desired immune responseagainst N. meningitidis infection. Preferred embodiments relate to amethod for the treatment, including amelioration, or prevention of N.meningitidis infection in a human comprising administering to a human animmunologically effective amount of the composition.

The phrase “immunologically effective amount,” as used herein, refers tothe administration of that amount to a mammalian host (preferablyhuman), either in a single dose or as part of a series of doses,sufficient to at least cause the immune system of the individual treatedto generate a response that reduces the clinical impact of the bacterialinfection. This may range from a minimal decrease in bacterial burden toprevention of the infection. Ideally, the treated individual will notexhibit the more serious clinical manifestations of the bacterialinfection. The dosage amount can vary depending upon specific conditionsof the individual. This amount can be determined in routine trials orotherwise by means known to those skilled in the art.

Another specific aspect of the present invention relates to using as theimmunogenic composition a vector or plasmid which expresses an proteinof this invention, or an immunogenic portion thereof. Accordingly, afurther aspect this invention provides a method of inducing an immuneresponse in a mammal, which comprises providing to a mammal a vector orplasmid expressing at least one isolated 2086 polypeptide. The proteinof the present invention can be delivered to the mammal using a livevector, in particular using live recombinant bacteria, viruses or otherlive agents, containing the genetic material necessary for theexpression of the polypeptide or immunogenic portion as a foreignpolypeptide.

According to a further implementation of the present invention, a methodis provided for diagnosing bacterial meningitis in a mammal comprising:detecting the presence of immune complexes in the mammal or a tissuesample from said mammal, said mammal or tissue sample being contactedwith an antibody composition comprising antibodies thatimmunospecifically bind with at least one polypeptide comprising theamino acid sequence of any of the even numbered SEQ ID NOS: 2-252;wherein the mammal or tissue sample is contacted with the antibodycomposition under conditions suitable for the formation of the immunecomplexes.

Viral and Non-Viral Vectors

Preferred vectors, particularly for cellular assays in vitro and invivo, are viral vectors, such as lentiviruses, retroviruses, herpesviruses, adenoviruses, adeno-associated viruses, vaccinia virus,baculovirus, and other recombinant viruses with desirable cellulartropism. Thus, a nucleic acid encoding a 2086 protein or immunogenicfragment thereof can be introduced in vivo, ex vivo, or in vitro using aviral vector or through direct introduction of DNA. Expression intargeted tissues can be effected by targeting the transgenic vector tospecific cells, such as with a viral vector or a receptor ligand, or byusing a tissue-specific promoter, or both. Targeted gene delivery isdescribed in PCT Publication No. WO 95/28494, which is incorporatedherein by reference in its entirety.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art (e.g., Millerand Rosman, BioTechniques, 1992, 7:980-990). Preferably, the viralvectors are replication-defective, that is, they are unable to replicateautonomously in the target cell. Preferably, the replication defectivevirus is a minimal virus, i.e., it retains only the sequences of itsgenome which are necessary for encapsulating the genome to produce viralparticles.

DNA viral vectors include an attenuated or defective DNA virus, such asbut not limited to herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, which entirely or almost entirely lack viralgenes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Molec. Cell. Neurosci., 1991, 2:320-330), defective herpes virusvector lacking a glyco-protein L gene, or other defective herpes virusvectors (PCT Publication Nos. WO 94/21807 and WO 92/05263); anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. (J. Clin. Invest., 1992, 90:626-630; seealso La Salle et al., Science, 1993, 259:988-990); and a defectiveadeno-associated virus vector (Samulski et al., J. Virol., 1987,61:3096-3101; Samulski et al., J. Virol., 1989, 63:3822-3828; Lebkowskiet al., Mol. Cell. Biol., 1988, 8:3988-3996), each of which isincorporated by reference herein in its entirety.

Various companies produce viral vectors commercially, including, but notlimited to, Avigen, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys(Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors), incorporatedby reference herein in its entirety.

Adenovirus Vectors.

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of this invention to a variety ofcell types. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to usingtype 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses ofanimal origin (see PCT Publication No. WO 94/26914). Those adenovirusesof animal origin which can be used within the scope of the presentinvention include adenoviruses of canine, bovine, murine (example: Mav1,Beard et al., Virology, 1990, 75-81), ovine, porcine, avian, and simian(example: SAV) origin. Preferably, the adenovirus of animal origin is acanine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan orA26/61 strain, ATCC VR-800, for example). Various replication defectiveadenovirus and minimum adenovirus vectors have been described (PCTPublication Nos. WO 94/26914, WO 95/02697, WO 94/28938, WO 94/28152, WO94/12649, WO 95/02697, WO 96/22378). The replication defectiverecombinant adenoviruses according to the invention can be prepared byany technique known to the person skilled in the art (Levrero et al.,Gene, 1991, 101:195; European Publication No. EP 185 573; Graham, EMBOJ., 1984, 3:2917; Graham et al., J. Gen. Virol., 1977, 36:59).Recombinant adenoviruses are recovered and purified using standardmolecular biological techniques, which are well known to persons ofordinary skill in the art.

Adeno-Associated Viruses.

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize that can integrate, in a stable and site-specific manner, into thegenome of the cells which they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The AAV genome has been cloned, sequenced andcharacterized. The use of vectors derived from the AAVs for transferringgenes in vitro and in vivo has been described (see, PCT Publication Nos.WO 91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368 and 5,139,941;European Publication No. EP 488 528). The replication defectiverecombinant AAVs according to the invention can be prepared bycotransfecting a plasmid containing the nucleic acid sequence ofinterest flanked by two AAV inverted terminal repeat (ITR) regions, anda plasmid carrying the AAV encapsidation genes (rep and cap genes), intoa cell line which is infected with a human helper virus (for example anadenovirus). The AAV recombinants which are produced are then purifiedby standard techniques.

Retrovirus Vectors.

In another implementation of the present invention, the nucleic acid canbe introduced in a retroviral vector, e.g., as described in U.S. Pat.No. 5,399,346; Mann et al., Cell, 1983, 33:153; U.S. Pat. Nos. 4,650,764and 4,980,289; Markowitz et al., J. Virol., 1988, 62:1120; U.S. Pat. No.5,124,263; European Publication Nos. EP 453 242 and EP178 220; Bernsteinet al., Genet. Eng., 1985, 7:235; McCormick, BioTechnology, 1985, 3:689;PCT Publication No. WO 95/07358; and Kuo et al., Blood, 1993, 82:845,each of which is incorporated by reference in its entirety. Theretroviruses are integrating viruses that infect dividing cells. Theretrovirus genome includes two LTRs, an encapsidation sequence and threecoding regions (gag, pol and env). In recombinant retroviral vectors,the gag, pol and env genes are generally deleted, in whole or in part,and replaced with a heterologous nucleic acid sequence of interest.These vectors can be constructed from different types of retrovirus,such as, HIV, MoMuLV (“murine Moloney leukaemia virus” MSV (“murineMoloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV (“spleennecrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus. Suitablepackaging cell lines have been described in the prior art, in particularthe cell line PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line(PCT Publication No. WO 90/02806) and the GP+envAm-12 cell line (PCTPublication No. WO 89/07150). In addition, the recombinant retroviralvectors can contain modifications within the LTRs for suppressingtranscriptional activity as well as extensive encapsidation sequenceswhich may include a part of the gag gene (Bender et al., J. Virol.,1987, 61:1639). Recombinant retroviral vectors are purified by standardtechniques known to those having ordinary skill in the art.

Retroviral vectors can be constructed to function as infectiousparticles or to undergo a single round of transfection. In the formercase, the virus is modified to retain all of its genes except for thoseresponsible for oncogenic transformation properties, and to express theheterologous gene. Non-infectious viral vectors are manipulated todestroy the viral packaging signal, but retain the structural genesrequired to package the co-introduced virus engineered to contain theheterologous gene and the packaging signals. Thus, the viral particlesthat are produced are not capable of producing additional virus.

Retrovirus vectors can also be introduced by DNA viruses, which permitsone cycle of retroviral replication and amplifies transfectionefficiency (see PCT Publication Nos. WO 95/22617, WO 95/26411, WO96/39036 and WO 97/19182).

Lentivirus vectors. In another implementation of the present invention,lentiviral vectors can be used as agents for the direct delivery andsustained expression of a transgene in several tissue types, includingbrain, retina, muscle, liver and blood. The vectors can efficientlytransduce dividing and nondividing cells in these tissues, and effectlong-term expression of the gene of interest. For a review, see,Naldini, Curr. Opin. Biotechnol., 1998, 9:457-63; see also Zufferey, etal., J. Virol., 1998, 72:9873-80). Lentiviral packaging cell lines areavailable and known generally in the art. They facilitate the productionof high-titer lentivirus vectors for gene therapy. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linethat can generate virus particles at titers greater than 106 IU/mL forat least 3 to 4 days (Kafri, et al., J. Virol., 1999, 73: 576-584). Thevector produced by the inducible cell line can be concentrated as neededfor efficiently transducing non-dividing cells in vitro and in vivo.

Non-Viral Vectors.

In another implementation of the present invention, the vector can beintroduced in vivo by lipofection, as naked DNA, or with othertransfection facilitating agents (peptides, polymers, etc.). Syntheticcationic lipids can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Felgner, et. al., Proc. Natl.Acad. Sci. U.S.A., 1987, 84:7413-7417; Felgner and Ringold, Science,1989, 337:387-388; see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A.,1988, 85:8027-8031; Ulmer et al., Science, 1993, 259:1745-1748). Usefullipid compounds and compositions for transfer of nucleic acids aredescribed in PCT Patent Publication Nos. WO 95/18863 and WO 96/17823,and in U.S. Pat. No. 5,459,127. Lipids may be chemically coupled toother molecules for the purpose of targeting (see Mackey, et. al.,supra). Targeted peptides, e.g., hormones or neurotransmitters, andproteins such as antibodies, or non-peptide molecules could be coupledto liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g., PCT PatentPublication No. WO 95/21931), peptides derived from DNA binding proteins(e.g., PCT Patent Publication No. WO 96/25508), or a cationic polymer(e.g., PCT Patent Publication No. WO 95/21931).

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for vaccine purposes or gene therapy can beintroduced into the desired host cells by methods known in the art,e.g., electroporation, microinjection, cell fusion, DEAE dextran,calcium phosphate precipitation, use of a gene gun, or use of a DNAvector transporter (e.g., Wu et al., J. Biol. Chem., 1992, 267:963-967;Wu and Wu, J. Biol. Chem., 1988, 263:14621-14624; Canadian PatentApplication No. 2,012,311; Williams et al., Proc. Natl. Acad. Sci. USA,1991, 88:2726-2730). Receptor-mediated DNA delivery approaches can alsobe used (Curiel et al., Hum. Gene Ther., 1992, 3:147-154; Wu and Wu, J.Biol. Chem., 1987, 262:4429-4432). U.S. Pat. Nos. 5,580,859 and5,589,466 disclose delivery of exogenous DNA sequences, free oftransfection facilitating agents, in a mammal Recently, a relatively lowvoltage, high efficiency in vivo DNA transfer technique, termedelectrotransfer, has been described (Mir et al., C. P. Acad. Sci., 1988,321:893; PCT Publication Nos. WO 99/01157; WO 99/01158; WO 99/01175).Accordingly, additional embodiments of the present invention relates toa method of inducing an immune response in a human comprisingadministering to said human an amount of a DNA molecule encoding a 2086polypeptide of this invention, optionally with atransfection-facilitating agent, where said polypeptide, when expressed,retains immunogenicity and, when incorporated into an immunogeniccomposition and administered to a human, provides protection withoutinducing enhanced disease upon subsequent infection of the human withNeisseria sp. pathogen, such as N. meningitidis.Transfection-facilitating agents are known in the art and includebupivicaine, and other local anesthetics (for examples see U.S. Pat. No.5,739,118) and cationic polyamines (as published in International PatentApplication WO 96/10038), which are hereby incorporated by reference.

The present invention also relates to an antibody, which may either be amonoclonal or polyclonal antibody, specific for 2086 polypeptides asdescribed above. Such antibodies may be produced by methods which arewell known to those skilled in the art.

Bacterial Expression Systems and Plasmids

This invention also provides a recombinant DNA molecule, such as avector or plasmid, comprising an expression control sequence havingpromoter sequences and initiator sequences and a nucleotide sequencewhich codes for a polypeptide of this invention, the nucleotide sequencebeing located 3′ to the promoter and initiator sequences. In yet anotheraspect, the invention provides a recombinant DNA cloning vehicle capableof expressing a 2086 polypeptide comprising an expression controlsequence having promoter sequences and initiator sequences, and anucleotide sequence which codes for a 2086 polypeptide, the nucleotidesequence being located 3′ to the promoter and initiator sequences. In afurther aspect, there is provided a host cell containing a recombinantDNA cloning vehicle and/or a recombinant DNA molecule as describedabove. Suitable expression control sequences and host cell/cloningvehicle combinations are well known in the art, and are described by wayof example, in Sambrook et al. (1989).

Once recombinant DNA cloning vehicles and/or host cells expressing adesired a polypeptide of this invention have been constructed bytransforming, transfecting or infecting such cloning vehicles or hostcells with plasmids containing the corresponding 2086 polynucleotide,cloning vehicles or host cells are cultured under conditions such thatthe polypeptides are expressed. The polypeptide is then isolatedsubstantially free of contaminating host cell components by techniqueswell known to those skilled in the art.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those skilled in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inview of the present disclosure, appreciate that many changes can be madein the specific embodiments which are disclosed and still obtain a likeor similar result without departing from the spirit and scope of theinvention.

EXAMPLES Example 1 Identification of a Neisserial Membrane ProteinExtract Capable of Eliciting Bactericidal Antibodies AgainstHeterologous Strains

Referring to Table II below, LOS-depleted outer membrane proteinpreparations have been shown to elicit bactericidal antibodies. Theseantibodies are often directed towards the PorA of the respective strain.LOS-depleted outer membrane preparations from serogroup B meningococcalstrain 8529 (B:15:P1.7b,3) are unusual in this manner because theyunexpectedly elicit bactericidal antibodies to several heterologousstrains.

TABLE II BC Activity of Anti-sOMPS Against Different Strains of N.meningitidis Anti-serum Week 6 H44/76 5315 H355 M982 880049 8529* NMBSerosubtype P1.7,16 P1.5 P1.15 P1.9 P1.4 P1.3 P1.5,2 sOMPs H44/76 25 μg1,000 <50 <50 <50 <50 980 <50 QS-21 20 μg sOMPs 5315 25 μg 50 <50 <50<50 <50 2170 <50 QS-21 20 μg sOMPs H355 25 μg <50 <50 450 <50 <50 860<50 QS-21 20 μg sOMPs M982 25 μg 92 <50 <50 300 <50 1100 <50 QS-21 20 μgsOMPs 880049 25 μg 50 <50 <50 <50 <50 1190 <50 QS-21 20 μg sOMPs 8529 25μg 1,000 <50 450 50 215 >4050 <50 QS-21 20 μg (81.7) sOMPs 2996 25 μg<50 <50 <50 <50 <50 790 148 QS-21 20 μg Whole-cell control 450 50 100500 150 >1350 952 serum 25 μg 3DMPL 25 μg (66.0)

To facilitate the isolation and characterization of the antigen(s)responsible for eliciting heterologous bactericidal antibodies, wesought to identify which detergent optimally extracted the antigen(s)

Strains and Culture Conditions.

N. meningitidis strain 8529 from a frozen vial was streaked onto a GCplate. (The meningococcal strain 8529 was received from The RIVM,Bilthoven, The Netherlands). The plate was incubated at 36C/5% CO₂ for7.5 h. Several colonies were used to inoculate a flask containing 50 mLof modified Frantz medium+GC supplement. The flask was incubated in anair shaker at 36° C. and agitated at 200 RPM for 4.5 h. 5 mL was used toinoculate a Fernbach flask containing 450 mL of modified Frantzmedium+GC supplement. The flask was incubated in an air shaker at 36° C.and agitated at 100 RPM for 11 h. The entire 450 mL was used toinoculate 8.5 L of modified Frantz medium+GC supplement in a 10 Lfermentor.

Composition of Modified Frantz Medium:

Glutamic acid 1.3 g/L Cysteine 0.02 Sodium phosphate, dibasic, 7 hydrate10 Potassium chloride 0.09 Sodium chloride 6 Ammonium chloride 1.25Dialyzed yeast extract (YE) 40 ml (25% YE soln. dialyzed agains 5volumes of dH₂O overnight, then autoclaved)GC supplement 100×, filter sterilize

GC supplement 100X, filter sterilize Dextrose 400 g/L Glutamic acid 10Cocarboxylase 0.02 Ferric nitrate 0.5

The following parameters were controlled during fermentation:Temperature=36° C.; pH=7.4; Dissolved Oxygen=20%. Several drops ofP-2000 antifoam were added to control foaming. The culture was grown tostationary phase. Cells were harvested by centrifugation at OD650=5.25.A total of 100-300 grams of wet cell paste is typically harvested from˜8.5 L of culture.

Partial Purification of Outer Membrane Protein Fractions fromMeningococci which Elicit Heterologous Bactericidal Antibodies:

100 gms wet weight of cells were suspended, to a volume five times thewet weight, with 10 mM HEPES-NaOH, pH 7.4, 1 mM Na2EDTA and lysed bypassage through a 110Y microfluidizer equipped with a chamber at 18,000psi. The cell lysate was clarified and the cell envelope isolated bycentrifugation at 300,000×g for 1 hour at 10° C. The cell envelopes werewashed 2× with the same buffer by suspension with a homogenizer followedby centrifugation as above. The cell envelopes were then extracted with320 mL of 1% (w/v) TRITON X-100 in 10 mM HEPES-NaOH, pH 7.4, 1 mM MgCl₂.Referring to Table III below, results from sequential differentialdetergent extraction using TRITON X-100 and ZWITTERGENT 3-14 followed byimmunization of mice, allowed us to determine that the TRITON extractsoptimally extracted the candidate(s) of interest. This TRITON X-100extract, eliciting bactericidal antibody response against 4 out of fivestrains listed in table III, was then fractionated by preparativeisoelectric focusing (IEF) in a BioRad Rotophor unit. Ampholyteconcentrations were 1% pH 3-10 mixed with 1% pH 4-6. As shown in TableIII, several fractions were found to elicit a heterologous bactericidalresponse. The fractions obtained from IEF, which focused in the pH rangeof 5.5-7.8, elicited a heterologous response to the most strains asdetermined by the bactericidal assay. The pooled IEF fractions wereconcentrated and the ampholytes removed by ethanol precipitation. Afurther purification was achieved by adsorbing some of the proteinsobtained in the pH range of about 5.5-7.8 on an anion exchange columnand comparing the bactericidal activity obtained after immunizing micewith the adsorbed and unadsorbed proteins. Referring again to Table II,while many proteins were adsorbed to the anion exchange resin, theproteins which were not adsorbed by the column elicited moreheterologous bactericidal antibodies.

TABLE III BC₅₀ Target Strain Method Fraction H44/76 880049 H355 539*M982 LOS- sOMPs 1,000 215 450 NC 50 depleted Detergent CytoplasmicExtract 200 NT NT NT NT Extractions TX-100 >800 >800 >800 >800 <25ZWITTERGENT 400 >25 100 400 <25 3-12 ZWITTERGENT <25 NT NT NT NT 3-14Zw.3-14 + NaCl <25 NT NT NT NT Sarcosyl <25 NT NT NT NT Zw.3-14 + heat<25 NT NT NT NT Preparative Fractions 1-3 50 NT NT NT NT IEF (pH2.3-3.9) Fraction 4 (pH 4.1) >800 <25 100 <25 NT Fraction 5 (pH4.3) >800 <25 100 200 NT Fraction 6 (pH 4.5) 400 NT NT NT NT Fraction 7(pH 4.8) <25 NT NT NT NT Fractions 8-9 <25 NT NT NT NT (pH 5.0-5.3)Fractions 10-17 >800 200 <800 <800 NT (pH 5.5-7.8) Anion Adsorbed 400 NT100 100 NT Exchange Unadsorbed >6,400 NT <800 <800 NT NT: not tested*Clinical isolate 539 is a homologous strain to 8529, isolated from thesame outbreak

As shown in FIG. 1A, two major proteins were present in the unadsorbedfraction as determined by SDS-PAGE. To identify these proteins, twotypes of analysis were performed. One analysis was to perform limitedproteolytic degradation (See FIG. 1A, and FIG. 1B) followed by isolationof peptides and direct protein sequencing. The other analysis was toperform SDS-PAGE followed by gel excision, proteolytic digestion, andLC-MS/MS (Liquid Chromotography tandem Mass Spectrometry), (see FIG. 3)to obtain mass spectral information on the components of thepreparations of interest. (See peptide mapping and sequencing methodsdescribed later in this section)

The N. meningitidis A Sanger genomic sequence was analyzed using themethods and algorithms described in Zagursky and Russell, 2001,BioTechniques, 31:636-659. This mining analysis yielded over 12,000possible Open Reading Frames (ORFs). Both the direct sequence data andthe mass spectral data described above indicated that the majorcomponents of the unadsorbed fraction were the products of several ORFspresent in an analysis of the Sanger database. The three predominantproteins identified by this methodology correspond to ORFs 4431, 5163and 2086, (see FIGS. 1B and 3).

Although ORF 4431 was the most predominant protein identified in thefractions, mouse antibodies to recombinant lipidated 4431 were notbactericidal and did not provide a protective response in an animalmodel. Additional analysis of ORF 5163 is in progress.

The second most predominant component of the preparations describedherein corresponds to the product of ORF 2086.

Immunogenicity Methods: Preparation of Antisera:

Except where noted, protein compositions/vaccines were formulated with25 μg of total protein and were adjuvanted with 20 μg QS-21. A 0.2 mLdose was administered by subcutaneous (rump) injection to 6-8 week oldfemale Swiss-Webster mice at week 0 and 4. Bleeds were collected at week0 and 4, and a final exsanguination bleed was performed on week 6.

Bactericidal Assay:

Bactericidal assays were performed essentially as described (SeeMountzouros and Howell, 2000, J. Clin. Microbiol. 38(8):2878-2884).Complement-mediated antibody-dependent bactericidal titers for the SBAwere expressed as the reciprocal of the highest dilution of test serumthat killed 50% of the target cells introduced into the assays (BC₅₀titer).

Methods Used to Identify 2086 Protein: Cyanogen Bromide Cleavage andDirect Sequencing of Fragments:

Cyanogen Bromide cleavage of Anion Exchange Unadsorbed Fraction (AEUF).The AEUF was precipitated with 90% cold ethanol and was solubilized with10 mg/mL cyanogen bromide in 70% formic acid to a protein concentrationof 1 mg/mL. The reaction was performed overnight at room temperature inthe dark. The cleaved products were dried down by speed vacuum, and thepellet was solubilized with HE/0.1% reduced TX-100. SDS-PAGE followed byN-terminal amino acid sequencing were used to identify the components ofthis fraction.

Protease Digestion/Reverse Phase/N-Terminal Sequencing to IdentifyComponents:

The AEUF was digested with either GluC (V8), LysC or ArgC. The proteinto enzyme ratio was 30 μg protein to 1 μg enzyme. The digestion wascarried out at 37° C. overnight. The digested protein mixture (30 μg)was passed over a seven micron Aquapore RF-300 column and was elutedwith a gradient of 10-95% acetonitrile in 0.1% trifluoroacetic acid, andpeaks were collected manually. A no protein blank was also run, and thepeaks from this were subtracted from the sample chromatogram. Peaksoccurring only in the sample run were analyzed by mass spectrometer, andthose samples giving a clear mass were analyzed for N-terminal aminoacid sequencing.

N-Terminal Amino Acid Sequencing:

For bands excised from a blot, the protein sample is transferred from anSDS gel to a PVDF membrane, stained with Amido Black (10% acetic acid,0.1% amido black in deionized water) and destained in 10% acetic acid.The desired protein band is then excised from all ten lanes using amethanol cleaned scalpel or mini-Exacto knife and placed in the reactioncartridge of the Applied Biosystems 477A Protein Sequencer. For directsequencing of samples in solution, the Prosorb cartridge is assembledand the PVDF wetted with 60 μL of methanol. The PVDF is rinsed with 50μL of deionized water and the sample (50 μL) is loaded to the PVDF.After 50 μL of deionized water is used to rinse the sample, the ProsorbPVDF is punched out, dried, and placed in the reaction cartridge of theApplied Biosystems 477A Protein Sequencer. For both methods, the AppliedBiosystems N-terminal Sequencer is then run under optimal blotconditions for 12 or more cycles (1 cycle Blank, 1 cycle Standard, and10 or more cycles for desired residue identification) and PTH-amino aciddetection is done on the Applied Biosystems 120A PTH Analyzer. Thecycles are collected both on an analog chart recorder and digitally viathe instrument software. Amino acid assignment is done using the analogand digital data by comparison of a standard set of PTH-amino acids andtheir respective retention times on the analyzer (cysteine residues aredestroyed during conversion and are not detected). Multiple sequenceinformation can be obtained from a single residue and primary versussecondary assignments are made based on signal intensity.

LC-MS/MS

Protein samples purified by IEF were further analyzed bySDS-polyacrylamide gel electrophoresis. Proteins were visualized byCOOMASSIE BLUE staining, and bands of interest were excised manually,then reduced, alkylated and digested with trypsin (Promega, Madison,Wis.) in situ using an automated in-gel tryptic digestion robot (1).After digestion, peptide extracts were concentrated to a final volume of10-20 μL using a Savant Speed Vac Concentrator (ThermoQuest, Holdbrook,NYH).

Peptide extracts were analyzed on an automated microelectrosprayreversed phase HPLC. In brief, the microelectrospray interface consistedof a Picofrit fused silica spray needle, 50 cm length by 75 um ID, 8 umorifice diameter (New Objective, Cambridge Mass.) packed with 10 um C18reversed-phase beads (YMC, Wilmington, N.C.) to a length of 10 cm. ThePicofrit needle was mounted in a fiber optic holder (Melles Griot,Irvine, Calif.) held on a home-built base positioned at the front of themass spectrometer detector. The rear of the column was plumbed through atitanium union to supply an electrical connection for the electrosprayinterface. The union was connected with a length of fused silicacapillary (FSC) tubing to a FAMOS autosampler (LC-Packings, SanFrancisco, Calif.) that was connected to an HPLC solvent pump (ABI 140C,Perkin-Elmer, Norwalk, Conn.). The HPLC solvent pump delivered a flow of50 μL/min which was reduced to 250 nL/min using a PEEK microtightsplitting tee (Upchurch Scientific, Oak Harbor, Wash.), and thendelivered to the autosampler using an FSC transfer line. The LC pump andautosampler were each controlled using their internal user programs.Samples were inserted into plastic autosampler vials, sealed, andinjected using a 5 μL sample loop.

Microcapillary HPLC-Mass Spectrometry:

Extracted peptides from in-gel digests were separated by themicroelectrospray HPLC system using a 50 minute gradient of 0-50%solvent B (A: 0.1M HoAc, B: 90% MeCN/0.1M HoAc). Peptide analyses weredone on a Finnigan LCQ ion trap mass spectrometer (ThermoQuest, SanJose, Calif.) operating at a spray voltage of 1.5 kV, and using a heatedcapillary temperature of 150° C. Data were acquired in automated MS/MSmode using the data acquisition software provided with the instrument.The acquisition method included 1 MS scan (375-1200 m/z) followed byMS/MS scans of the top 3 most abundant ions in the MS scan. The dynamicexclusion and isotope exclusion functions were employed to increase thenumber of peptide ions that were analyzed (settings: 3 amu=exclusionwidth, 3 min=exclusion duration, 30 secs=pre-exclusion duration, 3amu=isotope exclusion width). Automated analysis of MS/MS data wasperformed using the SEQUEST computer algorithm incorporated into theFinnigan Bioworks data analysis package (ThermoQuest, San Jose, Calif.)using the database of proteins derived from the complete genome of N.meningitidis (from Sanger). The results of the study are illustrated inFIG. 3.

Example 2 Cloning of Recombinant Lipidated P2086 (rLP2086) A.) NativeLeader Sequence: Source Materials:

The ORF 2086 gene was amplified by PCR from a clinical isolate of aserogroup B Neisseria meningitidis strain designated 8529. Theserogroup, serotype and serosubtype of this strain is shown inparentheses; 8529 (B:15, P1:7b,3). This meningococcal strain wasreceived from The RIVM, Bilthoven, The Netherlands. The mature 2086protein gene sequence from meningococcal strain 8529 is provided hereinas SEQ ID. NO. 212.

PCR Amplification and Cloning Strategy:

A visual inspection of ORF 2086 indicated that this gene had a potentiallipoprotein signal sequence. Additional analysis using a proprietaryHidden Markov Model Lipoprotein algorithm confirmed that ORF 2086contains a lipoprotein signal sequence. In order to recombinantlyexpress P2086 in a more native-like conformation, oligonucleotideprimers were designed to amplify the full length gene with thelipoprotein signal sequence intact and were based on an analysis of theSanger sequence for N. meningitidis A ORF 2086, _(5′ primer—CT ATT CTGCAT ATG ACT AGG AGC and 3′ primer—GCGC GGATCC TTA CTG CTT GGC GGC AAGACC), which are SEQ ID NO. 304 (Compound No. 4624) and SEQ ID NO. 303(Compound No. 4623), respectively (See also Table IV herein). The 2086gene was amplified by polymerase chain reaction (PCR) [ABI 2400 thermalcycler, Applied Biosystems, Foster City, Calif.] from N. meningitidisstrain 8529. The correct size amplified product was ligated and clonedinto pCR2.1-TOPO (Invitrogen). The plasmid DNA was restriction digestedwith NdeI and BamHI, gel purified and ligated into pET-27b(+) vector(Novagen).

Oligonucleotide primers described herein, were synthesized on aPerSeptive Biosystems oligonucleotide synthesizer, Applied Biosystems,Foster City Calif., using β-Cyanoethylphosphoramidite chemistry, AppliedBiosystems, Foster City Calif. The primers used for PCR amplification ofthe ORF 2086 gene families are listed in Table IV, which showsnon-limiting examples of primers of the present invention.

TABLE IV PRIMERS SEQ ID  NO. (Com- Restric- pound tion No.) PrimerSequence sites 303 Reverse GCGCGGATCCTTACTGCTTGGC BamHI (4623) GGCAAGACC304 Forward CTATTCTGCATATGACTAGGAGC NdeI (4624) 305 Forward AGCAGCGGAGGCGGCGGTGTC (4625) 306 Forward  TGCCGATGCACTAACCGCACC (5005)307 Reverse  CGTTTCGCAACCATCTTCCCG (5007) 308 Reverse GAGATCTCACTCACTCATTACT BglII (5135) GCTTGGCGGCAAGACCGATATG 309 Forward GCGGATCCAGCGGAGGGGGTGG BamHI (5658) TGTCGCC 310 Reverse GCGCATGCTTACTGCTTGGCGG SphI (5660) CAAGACCGATATG 311 Forward GCGGATCCAGCGGAGGCGGCGG BamHI (6385) AAGC 312 Forward GCGCAGATCTCATATGAGCAGC BglII and (6406) GGAGGGGGTGGTGTCGCCGCCG NdeIAYATWGGTGCGGGGCTTGCCG 313 Forward  CTATTCTGCGTATGACTAG (6470) 314Reverse  GTCCGAACGGTAAATTATCGTG (6472) 315 Forward GCGGATCCAGCGGAGGCGGCGG BamHI (6473) TGTCGCC 316 Forward GAGATCTCATATGAGCAGCGGA BglII and (6474) GGCGGCGGAAGC NdeI 317 Forward GACAGCCTGATAAACC (6495) 318 Reverse  GATGCCGATTTCGTGAACC (6496) 319Reverse  GCGCATGCCTACTGTTTGCCGG SphI (6543) CGATG 320 Reverse GAGATCTCACTCACTCACTACT BglII (6605) GTTTGCCGGCGATGCCGATTTC 321 Forward GCGCAGATCTCATATGAGCAGC BglII and (6721) GGAGGCGGCGGAAGCGGAGGCG NdeIGCGGTGTCACCGCCGACATAGG CACGrLP2086 Lipoprotein Expression Utilizing Native Leader Sequence:

Referring to FIG. 5, plasmid pPX7340 was transformed/transfected orinfected into BLR(DE3) pLysS host cells (Life Sciences). Onetransformant was selected and inoculated into 50 mL of Terrific Brothcontaining 2% glucose, kanamycin (30 μg/mL), chloramphenicol (30 μg/mL),and tetracycline (12 μg/mL). The OD600 for the overnight culture was6.0. The overnight culture was diluted out in 1 liter of Terrific Brothwith 1% glycerol and the same antibiotics. The starting OD600 was 0.4.After 2 hours the OD600 was 1.6 and a pre-induced sample was taken.Cells equivalent to an OD600=1 were centrifuged and the supernatant wasremoved. The whole cell pellet was resuspended in 150 μL Tris-EDTAbuffer and 150 μL of 2×SDS-PAGE sample buffer. IPTG was added to a finalconcentration of 1 mM. After 3.5 hours a post-induced sample was takenas described and analyzed on SDS-PAGE (See FIG. 4).

Purification of rLP2086:

The rLP2086 was solubilized from E. coli following differentialdetergent extraction. Unlike the P2086 in its native environment, therLP2086 was not significantly solubilized by TRITON X-100 or ZWITTERGENT3-12. The bulk of the rLP2086 was solubilized with sarcosyl, indicatingthat it interacts with the outer membrane components of E. colidifferently than it does in N. meningitidis. Once solubilized therLP2086 was purified similarly to the native protein in that many of thecontaminating E. coli proteins could be removed by adsorbtion to ananion exchange resin at pH 8. Despite being greater than one half a pHunit above its theoretical pI, the rLP2086 remained unadsorbed at pH 8.Further purification was achieved by adsorbtion of the rLP2086 to acation exchange resin at pH 4.5.

The homogeneity of the rLP2086 is shown in FIG. 2 following SDS-PAGE.The mass of rLP2086 was determined by MALDI-TOF mass spectral analysisto be 27,836. This mass differs from the theoretical mass of 27,100 by736, which approximates the mass of the N-terminal lipid modificationcommon to bacterial lipoproteins. Both native and rLP2086 appear to beouter membrane lipoproteins. Attempts with N-terminal sequencing wereblocked and this is consistent with the terminal modification.

Purification Methods:

Frozen pellets of BLR DE3 pLysS cells expressing P2086 were resuspendedin 10 mM HEPES-NaOH/1 mM EDTA/1 μg/mL Pefabloc SC protease inhibitor(Roche) pH 7.4 (HEP) at 20 mL/g wet cell weight and lysed bymicrofluidizer (Microfluidics Corporation Model 110Y). The cell lysatewas centrifuged at 150,000×g for one hour. The pellet was washed twicewith HEP and centrifuged twice, and the resulting membrane pellet wasfrozen overnight. The pellet was solubilized with 10 mM HEPES-NaOH/1 mMMgCl2/1% TX-100 pH 7.4 for 30 minutes, followed by centrifugation at150,000×g for 30 minutes. This was repeated three times. The membranepellet was washed as above twice with 50 mM Tris-HCl/5 mM EDTA/1%ZWITTERGENT 3-12 pH 8, followed by two washes each of 50 mM Tris-HCl/5mM EDTA/1% ZWITTERGENT 3-14 pH 8 and 50 mM Tris-HCl/5 mM EDTA/1%ZWITTERGENT 3-14/0.5M NaCl pH 8.

The rLP2086 was then solubilized with 50 mM Tris-HCl/5 mM EDTA/1%sarcosyl pH 8. This sarcosyl extract was adjusted to 1% ZWITTERGENT 3-14(Z3-14) and dialyzed twice against a 30 fold excess of 50 mM Tris-HCl/5mM EDTA/1% Z3-14. The dialyzed rLP2086 extract was precipitated with 90%ethanol to remove remaining sarcosyl, and solubilized with 50 mMTris-HCl/5 mM EDTA/1% Z3-14 pH 8 (TEZ). Insoluble material was removedby centrifugation, the supernatant was passed over an anion exchangechromatography column, and rLP2086 was collected in the unboundfraction. The unbound material was then dialyzed twice against a 30 foldexcess of 25 mM NaAc/1% Z3-14 pH 4.5, and passed over a cation exchangechromatography column. The rLP2086 was eluted with a 0-0.3M NaClgradient and analyzed by SDS-PAGE (COOMASSIE stain). The rLP2086 poolwas determined to be 84% pure by laser densitometry.

Surface Reactivity and Bactericidal Activity of Antisera to rLP2086Subfamily B.

Referring to Table VII, antisera to purified rLP2086 from the SubfamilyB strain 8529, demonstrated surface reactivity to all ten 2086 SubfamilyB strains tested by whole cell ELISA. Bactericidal activity was detectedagainst nine of ten 2086 Subfamily B strains expressing heterologousserosubtype antigens, PorAs. These strains are representative of strainscausing serogroup B meningococcal disease throughout western Europe, theAmericas, Australia, and New Zealand. The only strain which was notkilled in the bactericidal assay, 870227, reacted strongly with theanti-rLP2086 (Subfamily B) sera by whole cell ELISA, indicating thatthis strain expresses a protein with epitopes in common to P2086.

The 2086 Subfamily A strains listed in Table VII, were also tested forsurface reactivity by whole cell ELISA. Two out of three of thesestrains appeared to have a very low level of reactivity, indicating thatsome 2086 Subfamily A strains may not be cross-reactive with antibodiesraised to rLP2086 Subfamily B. The PCR amplification procedure used toidentify the 2086 Subfamily B gene from strain 8529 was also performedon strains 870446, NMB and 6557. No 2086 Subfamily B PCR amplifiedproduct was detected.

Immunogenicity Methods: Preparation of Antisera:

Vaccines were formulated as described previously in Example 1. However,a 10 μg dose was used.

Whole Cell Enzyme-Linked Immunosorbant Assay (ELISA):

N. meningitidis whole cell suspensions were diluted to an opticaldensity of 0.1 at 620 nm in sterile 0.01M phosphate, 0.137M NaCl, 0.002MKCl (PBS). From this suspension, 0.1 mL were added to each well of NUNCBACT 96 well plates (Cat#2-69620). Cells were dried on the plates atroom temperature for three days, then were covered, inverted and storedat 4° C. Plates were washed three times with wash buffer (0.01MTris-HCl, 0.139M NaCl/KCl, 0.1%dodecylpoly(oxyethylereneglycolether)_(n) n=23 (BRIJ-35g, available fromICI Americas, Inc., Wilmington, Del.), pH 7.0-7.4). Dilutions ofantisera were prepared in PBS, 0.05% TWEEN-20/Azide and 0.1 mL wastransferred to the coated plates. Plates were incubated for two hours at37° C. Plates were washed three times in wash buffer. Goat-anti-mouseIgG AP (Southern Biotech) was diluted at 1:1500 in PBS/0.05% TWEEN-20,0.1 mL was added to each well, and plates were incubated at 37° C. fortwo hours. Plates were washed (as above). Substrate solution wasprepared by diluting p-nitrophenyl phosphate (Sigma) in 1Mdiethanolamine/0.5 mM MgCl₂ to 1 mg/mL. Substrate was added to the plateat 0.1 mL per well and incubated at room temperature for one hour. Thereaction was stopped with 50 μL/well of 3N NaOH and plates were read at405 nm with 690 nm reference.

B.) P4 Leader Sequence: PCR Amplification and Cloning Strategy:

In order to optimize rLP2086 expression, the 2086 gene was cloned behindthe P4 signal sequence of nontypable Haemophilus influenzae (Green etal., 1991). Primers utilized for lipoprotein cloning are listed in TableIV and are identified by compound numbers: 5658, 5660, 6473, 6543 and6385. ORF 2086 was amplified from N. meningitidis B strain 8529 usingprimers with the following compound numbers 5658 and 5660. ORF 2086 wasamplified from N. meningitidis serogroup B strain CDC1573 using primerswith the following compound numbers 6385 and 5660. ORF 2086 wasamplified from N. meningitidis serogroup B strain 2996 using primerswith the following compound numbers 6473 and 6543. The N-terminal (5′)primers were designed to be homologous to the mature region of the 2086gene (starting at the serine residue at amino acid position 3 justdownstream of the cysteine). The restriction site BamHI (GGATTC) wasincorporated into the 5′ end of each N-terminal primer and resulted inthe insertion of a glycine residue in the mature protein at amino acidposition 2. The C-terminal (3′) primers were designed to be homologousto the C-terminal end of the 2086 gene and included the Stop codon aswell as an SphI site for cloning purposes. The amplified fragment fromeach N. meningitidis B strain was cloned into an intermediate vector andscreened by sequence analysis.

Plasmid DNA from correct clones was digested with BamHI and SphIrestriction enzymes (New England Biolabs, (NEB)). A vector designatedpLP339 (supplied by applicants' assignee) was chosen as the expressionvector. This vector utilizes the pBAD18-Cm backbone (Beckwith et al.,1995) and contains the P4 lipoprotein signal sequence and P4 gene ofnontypable Haemophilus influenzae (Green et al., 1991). The pLP339vector was partially digested with the restriction enzyme BamHI and thensubjected to SphI digestion. The amplified 2086 fragments (BamHI/SphI)were each ligated separately into the pLP339 vector (partialBamHI/SphI). This cloning strategy places the mature 2086 gene behindthe P4 lipoprotein signal sequence. The BamHI site remains in thecloning junction between the P4 signal sequence and the 2086 gene (Seethe plasmid construct shown in FIG. 7). The following is an example ofthe sequence at the BamHI cloning junction:

-   -   [P4 signal sequence]—TGT GGA TCC—[remaining 2086 mature nucleic        acid sequence]    -   [P4 signal sequence]—Cys Gly Ser—[remaining 2086 mature amino        acid sequence]

Referring to FIG. 7, each amplified fragment was cloned into a modifiedpBAD18-Cm vector containing the P4 leader sequence. Fermentation wasperformed on recombinant E. coli BLR pPX7343 which expresses rP4LP2086(recombinant P4 lipidated 2086) to try to increase the cell density byadding additional glucose. The fermentor was filled with 10 L completeM9 Minimal medium, according to Sambrook, supplemented with 1% glucose.

The initial concentration of glucose in the fermentor was 45 g/L. Thefermentor was inoculated to initial OD of −0.25. At ˜OD 25, additional20 g/L glucose was added. The culture was induced with 1% arabinose atglucose depletion at OD 63.4. The fermentation continued until 3 hoursafter induction. Samples were saved at t=0, 1, 2, 3 post induction andprotein quantified using BSA. At t=3, protein yield is ˜0.35 g/L, and 7%total cellular protein. A total of 895 grams of wet cell paste washarvested from ˜10 L of culture.

Purification of the rP4LP2086 was performed using the same methods asdescribed above in Example 2, section A.

Example 3 Development Genetics for Non-Lipidated Mature 2086 Protein

To further evaluate the immunogenicity of the 2086 protein, cloning andexpression of the non-lipidated form of P2086 were performed.

PCR Gene Amplification of the ORF 2086:

Oligonucleotides used for PCR amplification of the non-lipidated 2086gene are listed in the primer table, Table IV. The 2086 gene from strain8529 can be amplified with primers identified by compound numbers 5135and 6406 (SEQ ID NOS. 308 and 312, respectively), as indicated in thetable. The 2086 gene from strain CDC1573 can be amplified with primersidentified by compound numbers 5135 and 6474 (SEQ ID NOS. 308 and 316,respectively). The 2086 gene from strain 2996 can be amplified withprimers identified by compound numbers 6406 and 6605 (SEQ ID NOS. 312and 320, respectively).

Features of these primers include, a synthetic BglII restriction site ineach primer, a synthetic NdeI restriction site in compound numbers 6406and 6474 and termination codons in all three reading frames are presentin compound numbers 5135 and 6605. Primer numbers 6406 and 6474 amplifythe 2086 gene with an ATG (Met) fused to the second amino terminal codon(ACG) representing a single amino acid substitution (replaces TGC Cys)of the mature 2086 polypeptide.

The PCR cloning vector was TOPO-PCR2.1, Invitrogen, Valencia, Calif.

The vector used to express non-lipidated 2086 protein was pET9a fromNovagen, Madison, Wis.

The E. coli cloning strain was Top10, Invitrogen, Carlsbad, Calif.

The E. coli expression strain was BLR(DE3)pLysS, Novagen, Madison, Wis.

The culture media for cloning purposes was Terrific Broth liquid oragar, according to Sambrook et al., with 1% sterile glucose substitutedfor glycerol, and the appropriate antibiotic (ampicillin or kanamycin).

Plasmid purification was with Qiagen Spin Miniprep Kit (Valencia,Calif.).

Preparation of the Production Strain or Cell Line for Non-Lipidated 2086Expression:

The 2086 gene was amplified by polymerase chain reaction (PCR) [AmpliTaqand ABI 2400 thermal cycler, Applied Biosystems, Foster City, Calif.]from chromosomal DNA derived from meningococcal strain 8529. The PCRamplification of the 2086 gene utilized two oligonucleotide primers ineach reaction identified by compound numbers 6474 and 5135 (SEQ ID NOS.316 and 308, respectively). The amplified 2086 PCR product was cloneddirectly into the TOPO-PCR2.1 cloning vector and selected on TerrificBroth agar supplemented with 100 μg/m1 ampicillin and 20 μg/ml X-Gal.White colonies were selected and grown. Plasmid DNA was prepared using aQiagen miniprep kit and the plasmids were screened for the PCR fragmentinsert. PCR insert plasmids were subjected to DNA sequencing (Big Dyechemistry on an ABI377 sequencer, Applied Biosystems, Foster City,Calif.).

Plasmids exhibiting the correct DNA sequence were digested with BglIIrestriction enzyme and the BglII fragment was gel purified using aGeneClean II purification kit (Bio101, Carlsbad, Calif.). The purifiedBglII fragment was cloned into the BamHI site of the expression vectorpET9a. The pET9a/2086 clones were selected on Terrific Broth platessupplemented with 30 μg/ml kanamycin. Kanamycin resistant clones weregrown and miniprep plasmid DNA was prepared. The plasmids were screenedfor the appropriate orientation of the 2086 gene in the BamHI site.Correctly oriented plasmids represent a fusion of the T7-antigen to theamino terminus of 2086 gene (rP2086T7). These rP2086T7 gene fusions weretransformed into BLR(DE3)pLysS, selected on Terrific Broth/Kan plates,grown in Terrific Broth and induced to express the rP2086T7 fusionprotein with 1 mM IPTG (isopropyl β-D-thiogalactopyranoside). TherP2086T7 fusion protein expressed at high levels.

These fusion plasmids were then subjected to a NdeI restriction digest,which deletes the T7-antigen and links the mature 2086 gene directly tothe ATG start provided by the vector. These NdeI deleted plasmids weretransformed into Top10 cells and selected on Terrific Broth/Kan plates.Candidate clones were grown and miniprep plasmid DNA was prepared. Theplasmid DNA was subjected to DNA sequencing to confirm the deletion andthe integrity of the 2086 gene sequence. These plasmids are representedby the plasmid map designated pPX7328 (FIG. 6). Plasmids representingthe correct DNA sequence were transformed into BLR(DE3)pLysS, selectedon Terrific Broth/Kan plates, grown in Terrific Broth and induced toexpress the 2086 protein with IPTG. The pET9a vector failed to expressthe mature 2086 protein, in strain BLR(DE3)pLysS, when the T7-Tag wasremoved.

Production of Non-Lipidated 2086 Protein:

Purified plasmid DNA was used to transform the expression strainBLR(DE3)pLysS. BLR(DE3)pLysS cells carrying the plasmids are resistantto kanamycin and can be induced to express high levels of PorA proteinby the addition of 1 mM IPTG. The rP2086T7 fusion protein can beexpressed as insoluble inclusion bodies in the E. coli cell lineBLR(DE3)pLysS at ˜40% of total protein. This purified fusion protein wasused to immunize mice and generated significant levels of bactericidalantibodies against a heterologous meningococcal strain. (See Table V)

2086 Non-Lipidated Gene Mutagenesis:

PCR primer mutagenesis was performed on the 5′ end of the 2086 gene.Expression studies are under way to determine if the T7-Tag can beremoved while exhibiting the high expression levels of mature rP2086T7.

Purification of Non-Lipidated rP2086T7:

E. coli BLR(DE3)pLysS cells expressing non-lipidated rP2086T7 were lysedby microfluidizer in 10 mM Hepes-NaOH/5 mM EDTA/1 mM Pefabloc SC pH 7.4.The cell lysate was then centrifuged at 18,000×g for 30 minutes. Theinclusion body pellet was washed three times with 50 mM Tris-HCl/5 mMEDTA/1% TRITONX-100 pH 8 followed by centrifugation each time at24,000×g for 30 min. The inclusion body pellet was then washed twicewith 50 mM Tris-HCl/5 mM EDTA/1% ZWITTERGENT 3-14 pH 8 followed bycentrifugation each time at 24,000×g for 15 min. The inclusion bodypellet was then solubilized with 50 mM Tris-HCl/5 mM EDTA/4M Urea pH 8for two hours followed by centrifugation to remove insoluble material.The supernatant (solubilized rP2086T7) was split into four equalsamples. One sample was adjusted to 50 mM Tris-HCl/5 mM EDTA/250 mMNaCl/2M Urea pH8 (no detergent), one was adjusted to 50 mM Tris-HCl/5 mMEDTA/250 mM NaCl/2M Urea/1% hydrogenated TRITON X-100 pH8 (TX-100), onewas adjusted to 50 mM Tris-HCl/5 mM EDTA/250 mM NaCl/2M Urea/1%ZWITTERGENT 3-12 pH8 (Z3-12), and one was adjusted to 50 mM Tris-HCl/5mM EDTA/250 mM NaCl/2M Urea/1% ZWITTERGENT 3-14 pH8 (Z3-14) using stocksolutions. To remove the urea, samples were dialyzed to completionagainst the respective buffer containing no urea. The samples were thendialyzed to completion against the respective buffer containing no ureaand 60 mM NaCl to reduce the NaCl concentration. Insoluble material wasremoved by centrifugation at 2,000×g for 15 minutes, and the resultingsupernatant (refolded rP2086T7) was used for further experiments.Homogeneity of rP2086T7 was found to be 91-95% as determined usingCOOMASSIE stained SDS-PAGE and laser densitometry.

Immunogenicity Procedure—as Described in Example 2

This purified fusion protein was used to immunize mice and generatedsignificant levels of bactericidal antibodies against a heterologousmeningococcal strain. (See Table V below):

TABLE V Bactericidal titers of mouse antibody raised to rP2086T7HETEROLOGOUS MOUSE SERUM DESCRIPTION STRAIN/H44/76 AF780 week 6 r2086T7,10 ug 3200 Week 0 pool Pre-immune serum 10 AE203 week 6 rLP2086, 10 ug(positive 6400 control)* (*positive control sera generated byimmunization of mice with rLP2086)

Example 4 Development of Chimeric Clones of ORF 2086

The N-terminal region of the 2086 gene from strain CDC-1573 contains arepeated segment not present in the 2086 gene from strains 8529 and 2996(see FIG. 8). It appears that this repeated segment is responsible forincreased levels of recombinant 2086 protein expression from two E. colibased expression systems (pET and pBAD). The recombinant proteinexpression level from the CDC-1573 2086 gene was significantly better inthe pET and pBAD expression systems as compared to the recombinantexpression levels from the 2086 gene with strains 8529 and 2996 usingthe same systems. The N-terminal region of the 2086 gene from all threestrains is relatively homologous, except for this repeated segment.Therefore, it is reasonable to assume that by fusing the CDC-1573N-terminus to the 2086 genes from strains 8529 and 2996, that therecombinant 2086 protein levels expressed from these genes will increasewhen using the pET and pBAD systems.

Materials and Methods:

Chromosomal DNA from strains 8529 and 2996 was purified and used as atemplate for PCR amplification of the chimeric 2086 gene. PCR primerswith the compound numbers 6721 and 5135 (SEQ ID NOS. 321 and 308,respectively) were used to amplify the chimeric 2086 gene from strain8529 and PCR primers with the compound numbers 6721 and 6605 (SEQ IDNOS. 321 and 320, respectively) were used to amplify the chimeric 2086gene from strain 2996. The PCR products were cloned directly into thePCR2.1 TOPO vector from Invitrogen and then screened by DNA sequenceanalysis to identify an intact chimeric 2086 gene. That gene was thencleaved from the PCR2.1 vector with BglII and the BglII fragment wasinserted into the BamHI site of the pET9a plasmid. Plasmid inserts werescreened for the appropriate orientation and then subjected to a NdeIdigestion. The linear NdeI fragments were self-ligated to achieve thedeletion of a small NdeI fragment containing the T7-tag sequencecontributed by the pET9a vector. This deletion directly links the T7promoter to the 5′ end of the chimeric 2086 gene. The NdeI deletedplasmid was transformed into E. coli strain BL21(DE3) and kanamycinresistant colonies were screened for chimeric 2086 protein expressionwith IPTG induction.

Initial studies indicate that the chimeric 2086 gene from strain 2996expresses about twice as much recombinant protein as compared to thenative 2996/2086 gene when expressed in the pET9a system. The pBADsystem has not been tested yet.

Although only one experiment has been performed, the data indicate thatthere is an enhanced utility from the chimeric 2086 gene. The generationof CDC-1573 N-terminal fusions to the 2086 genes from strains 8529 and2996 provides enhanced recombinant 2086 protein expression.

Example 5 2086 PCR Screening of N. meningitidis Strains

In order to determine the conservation of the 2086 gene among clinicalisolates, PCR amplification was performed on 88 N. meningitidis strains.

Initial PCR identification of ORF 2086 utilized primers listed in TableIV (see Example 2 above) identified by compound numbers: 4623, 4624 and4625 (SEQ ID NOS. 303, 304 and 305, respectively). These primers weredesigned based on Sanger's N. meningitidis serogroup A sequence. Tofacilitate screening a large number of strains, internal primers weredesigned for the 2086 gene. A total of 88 N. meningitidis strains werescreened by PCR with the newly designed internal 2086 primers identifiedby compound numbers 5005 and 5007 (SEQ ID NOS. 306 and 307). With theseprimers the applicants were able to identify the 2086 gene from 63 ofthe 88 (−70%) N. meningitidis strains, (see Table VI-A).

Expanded regions surrounding the 2086 gene in Sanger's N. meningitidisserogroup A sequence and TIGR's N. meningitidis serogroup B sequencewere examined and aligned. Primers were designed to correspond toregions upstream and downstream of the 2086 gene. The purpose was toutilize these primers to amplify greater than full length 2086 genesfrom a variety of N. meningitidis strains for sequence comparison. PCRamplification of one strain (6557), using Compound Nos. 6470 and 6472(SEQ ID NOS: 313 and 314, respectively), resulted in a low yield ofproduct. The strain 6557 amplified product was cloned and plasmid DNAwas submitted for sequence analysis. Results indicated a new type of2086 gene with greater sequence variability than had previously beenseen. The 2086 gene from strain 6557 was ˜75% identical at the aminoacid level to the other strains sequenced. Interestingly, strain 6557was one of the 30% of strains that had previously tested negative by2086 PCR screening described above.

Internal primers specific to the C-terminal variable regions withinstrain 6557 were designed. These primers were used to screen for themore variable 2086 gene in the ˜30% of strains that had previouslytested negative by 2086 PCR screening. All available N. meningitidisstrains (n=88) were screened by PCR with these newly identified internal2086 primers (identified by compound numbers 6495 and 6496; SEQ ID NOS.159 and 160, respectively). Only the ˜30% of N. meningitidis strainsthat had previously tested negative by PCR for 2086 were PCR positive inthis screen. The set of genes amplified from the previously PCR negative(˜30%) strains should represent a new type of 2086 gene or a secondfamily of 2086 genes and herein are designated 2086 Subfamily A. The setof 2086 genes amplified from the ˜70% of strains with the 8529 derivedprimers are herein designated Subfamily B.

Subfamily A of 2086 genes is exemplified by the odd numbered SEQ IDNOS:1-173 without limitation. Subfamily B of 2086 genes is exemplified,without limitation, by the odd numbered SEQ ID NOS: 175-251

N. meningitidis strains used for PCR amplification studies were selectedfrom the following tables, Table VI-A and Table VI-B. The strains listedin the tables are provided as examples of N. meningitidis strains,without limitation. The strains listed in Table VI-A are classified in2086 protein Subfamily A and the strains listed in Table VI-B areclassified in 2086 protein Subfamily B. The strains listed in each tableare grouped by serosubtype. The strains are available from the followingfour sources as indicated in the table: MPHL-Manchester Public HealthLaboratory, Manchester, UK; RIVM, Bilthoven, The Netherlands; Universityof Iowa, College of Medicine, Department of Microbiology, Iowa City,Iowa; and Walter Reed Army Institute of Research, Washington, D.C.

TABLE VI-A Strain Serosubtype Source M97 251854 B:4z, PI:4 MPHL M98250622 B:2b, PI:10 MPHL M98 250572 B:2b, PI:10 MPHL M98 250771 B:4z,PI.22,14 MPHL M98 250732 B:4z, PI.22,14a MPHL M98 250809 B:15, PI:7,16MPHL M97 252697 B:1, PI:6, P1.18,25 MPHL M97 252988 B:4, PI:6,P1.18,25,6 MPHL M97 252976 B:4, PI:6, P1.18,25 MPHL M97 252153 B:4,PI:6, P1.18,25 MPHL M97 253248 B:15, PI:7, NT, 16 MPHL CDC1610 P1:NT4(15), P1.18-7,16-14 CDC CDC1521 P1.6,3 2b(4) CDC CDC1034 P1.7 4(15) CDCL8 P1.7,1 15(4) Walter Reed CDC1492 P1.7,1 4(15) CDC 870446 P1.12a,13RIVM CDC2369 P1.(9),14 CDC 6557 P1.(9),14, P1.22a,14a RIVM 2996 P1.5,2,P1.5a,2c RIVM NmB P1.5,2, P1.5a,2c UIOWA L3 P1.5,2 Walter Reed B16B6P1.5,2 RIVM CDC1135 CDC L5 P1.NT, P1.21-6,1 Walter Reed L4 P1.21,16Walter Reed W135 Walter Reed C11 C:16,P1.7,1 CDC Y Walter Reed

TABLE VI-B Strain Serosubtype Source M98 250670 B:1, PI:4 MPHL M98250024 B:1, PI:4 MPHL M97 253524 B:1, PI:4 MPHL M97 252060 B:1, PI:4MPHL M97 251870 B:4z, PI:4 MPHL M97 251836 B:4z, PI:4 MPHL M97 251830B:4z, PI:4 MPHL M97 251905 B:4z, PI:4 MPHL M97 251898 B:4z, PI:4 MPHLM97 251885 B:4z, PI:4 MPHL M97 251876 B:4z, PI:4 MPHL M97 251994 B:4z,PI:4 MPHL M97 251985 B:4z, PI:4 MPHL M97 251957 B:4z, PI:4 MPHL M97251926 B:4z, PI:4 MPHL M97 252045 B:4z, PI:4 MPHL M97 252038 B:4z, PI:4MPHL M97 252026 B:4z, PI:4 MPHL M97 252010 B:4z, PI:4 MPHL M97 252098B:4z, PI:4 MPHL M97 252083 B:4z, PI:4 MPHL M97 252078 B:4z, PI:4 MPHLM98 250735 B:4z, PI:15 MPHL M98 250797 B:4z, PI:15 MPHL M98 250768 B:4z,PI:15 MPHL M98 250716 B:2b, PI:10 MPHL M98 250699 B:4z,PI:10 MPHL M98250393 B:4z,PI:10 MPHL M98 250173 B:4z,PI:10 MPHL M97 253462 B:4z, PI:14MPHL M98 250762 B:15, PI:7,16 MPHL M98 250610 B:15, PI:7,16 MPHL M98250626 B:15, PI:7,16 MPHL M97 250571 B:15, PI:16 MPHL M97 252097 B:15,PI:16, P1.7b,16 MPHL M97 253092 B:1, PI:6 MPHL M97 252029 B:15,PI:7, NTMPHL M97 251875 B:15,PI:7, NT MPHL CDC1127 PI.7,16 4(15) CDC CDC982PI.7,16 4(15) CDC CDC1359 PI.7,16 4(15) CDC CDC798 PI.7,16 15(4) CDCCDC1078 PI.7,16 15(4) CDC CDC1614 PI.7,16 15(4) CDC CDC1658 PI.7,1615(4) CDC H44/76 PI.7,16 15(4) RIVM CDC1985 P1.7,13 4(15) CDC L6 P1.7,1?(4) Walter Reed CDC1573 P1.7.1 4(15) CDC L7 P1.7,(9),1 Walter ReedCDC937 P1.7,3, P1.7b,3 CDC 8529 P1.7,3, P1.7b,3 RIVM 880049 P1.7b,4 RIVMCDC2367 P1.15 4(15) CDC H355 P1.19,15 RIVM CDC1343 P1.14 4(15) CDC M982P1.22,9 RIVM 870227 P1.5c,10 RIVM B40 P1.5c,10 RIVM 5315 P1.5c,10 RIVMCDC983 P1.5,2 CDC CDC852 P1.5,2 CDC 6940 P1.18,25 (6) RIVM A4Other strains are readily available as isolates from infectedindividuals.

Example 6 Reactivity of rLP2086 Antisera Against Meningococcal Strains

The following table, Table VII, shows the cross-reactive and crossprotection capacity of the rLP2086 as described above. As indicated inthe table, the rLP2086 was processed and analyzed using a variety oftechniques including whole cell ELISA (WCE) titers, bactericidal assay(BCA) and Infant Rat (IR) assays to determine the bacterial cell surfacereactivity of a polyclonal antibody raised against the 2086 protein.

TABLE VII Reactivity of rLP2086-8529 antisera against multiplemeningococcal strains Strain Serosubtype WCE BC IR 2086 Subfamily A870446 P1.12a,13 808,615 >800 NmB P1.5a,2c 47,954 <100 6557 P1.22a,14a169,479 <25 − 2086 Subfamily B 880049 P1.7b,4 1,402,767 100 + H44/76P1.7,16 8,009,507 >6400 H355 P1.19,15 10,258,475 3,200 + 6940P1.18,25(6) 5,625,410 800 870227 P1.5c,10 4,213,324 <25 + 252097P1.7b,16 10,354,512 >800 539/8529 P1.7b,3 11,635,737 3,200 M982 P1.22,91,896,800 800 CDC-1573 P1.7a,1 208,259 25 CDC-937 P1.7b,(3)9,151,863 >800 + greater than 10 fold reduction in bacteremia − lessthan 10 fold reduction in bacteremia

Example 7

Various constructs for expressing ORF2086 protein were prepared. Thefollowing table, Table VIII, is an r2086 construct table which isprovided for the purpose of showing examples and illustrating animplementation of the present invention, without limitation thereto.

TABLE VIII r2086 Construct Summary Construct Promoter Leader ExpressionExtraction Vector % total Protein pPX7340 T7 native COOMASSIE sarcosylpET27b 2.5% processed soluble lipoprotein pPX7341 T7 P4 COOMASSIEsarcosyl pET27b 5% processed soluble lipoprotein pPX7343 Arabinose P4COOMASSIE sarcosyl pBAD18 cm 7-10% processed soluble lipoprotein pPX7325T7 T7-tag COOMASSIE inclusion pET9a 40-50% mature fusion/ bodies proteinmature pPX7328 T7 mature COOMASSIE soluble pET9a 10% mature protein

Example 8

Further studies with LOS depleted outer membrane proteins identifiedadditional strains producing outer membrane protein(s) other than PorAwhich were capable of eliciting bactericidal antibodies to strainsexpressing heterologous serosubtypes. The following describes furtherstudies to identify additional proteins according to one embodiment ofthe present invention, and specifically outer membrane lipoproteins,which can reduce the number of proteins required in a meningococcalimmunogenic composition. These further studies supplement the studiesdescribed in the previous examples.

Subcellular fractionation, differential detergent extraction,isoelectric focusing, and ion exchange chromatography were used inconjunction with immunization and bactericidal assays against multiplestrains to identify small groups of proteins of interest. Directsequencing of the main components indicated that the N-termini wereblocked. Internal protein sequences were obtained by direct sequencingof polypeptides derived from chemical and proteolytic digests. Thegenomic sequence of a group A meningococcal strain was downloaded fromthe Sanger Center and analyzed by our Bioinformatics group usingexisting and proprietary algorithms to create a searchable database. Thepeptide sequence data indicated that ORF2086 was of interest. Primersbased on this orf were used to PCR the P2086 gene from strain 8529.Analysis of the gene sequence, the fact that the N-terminus was blocked,and its subcellular location indicated that P2086 is a lipidated outermembrane protein (LP2086). rLP2086-8529 and variants from othermeningococcal strains were recombinantly expressed as lipoproteins in E.coli using the H. influenzae P4 signal sequence. These recombinantproteins were isolated from E. coli membranes by differential detergentextraction, purified using ion exchange chromatography, and used toimmunize mice. Mouse anti-LP2086 sera were able to facilitatebactericidal activity against several different serosubtype strains ofNmeningitidis. Further analysis of the P2086 genes from many N.meningitidis strains showed that these sequences fell into two groupsdesignated Subfamily A and Subfamily B. (See FIG. 12) The antiseraraised against the Subfamily B proteins were bactericidal against ninestrains expressing Subfamily B proteins, and one strain expressing aSubfamily A protein. Subfamily A antisera were bactericidal againstSubfamily A strains. A mixture of one rPorA and one rLP2086 elicitedcomplementary antibodies extending vaccine coverage beyond that inducedby either protein alone.

These observations lead to the following conclusions. rLP2086 antigensare capable of eliciting bactericidal antibodies against meningococcalstrains expressing heterologous PorAs and heterologous P2086 proteins.The P2086 family of antigens may be a useful vaccine or immunogeniceither alone or in combination with other neisserial antigens.

The following describes the foregoing study in detail. A complex mixtureof soluble outer membrane proteins (sOMPs) was found to elicit PorAindependent bactericidal antibody against strains expressingheterologous PorA proteins. A process of differential detergentextraction, isoelectric focusing and ion exchange chromatographyfollowed by mouse immunization was used to follow the immunologicallyactive components.

At each step, sera was assayed for surface reactivity and bactericidalactivity against several strains containing serosubtype antigens thatare representative of the worldwide epidemiology of meningococcaldisease.

This process of separation and immunization was used to identify a novelcross-reactive immunogenic candidate for Group B N. meningitidis.

Generation of PorA deficient strains—The porA chromosomal locus wascloned into plasmid pPX7016 from strain 2996. Within the plasmid theporA promoter, the S/D box and the first 38 N-terminal codons have beendeleted and replaced with a self contained KanR expressing cassette. Theplasmids were linearized with restriction enzymes and naturallytransformed into the serosubtype strains PI:5,2; PI:9; PI:7,16; PI:15;PI:4; PI:3 & PI:10. Kanamycin resistant transformants were selected andscreened for the loss of PorA by serosubtype specific monoclonals in anELISA.

Bactericidal Assay: See Mountzourous, K. T. and Howell, A. P. Detectionof Complement-Mediated Antibody-Dependent Bactericidal Activity in aFlourescence-Based Serum Bactericidal Assay for Group B Neisseriameningitidis. J Clin Microbiol. 2000; 38:2878-2884.

Whole Cell Enzyme Linked Immonosorbant Assay (ELISA): N. meningitidiswhole cell suspensions were diluted to an optical density of 0.1 at 620nm in sterile 0.01M phosphate, 0.137M NaCl, 0.002M KCl (PBS). From thissuspension, 0.1 mL were added to each well of NUNC BACT 96 well plates(Cat#2-69620). Cells were dried on the plates at 37° C. overnight, thenwere covered, inverted and stored at 4° C. Plates were washed threetimes with wash buffer (0.01M Tris-HCl, 0.139M NaCl/KCl, 0.1% BRIJ-35,pH 7.0-7.4). Dilutions of antisera were prepared in PBS, 0.05%TWEEN-20/Azide and 0.1 mL was transferred to the coated plates andincubated for two hours at 37° C. Plates were washed three times in washbuffer. Goat-anti-mouse IgG AP (Southern Biotech) was diluted at 1:1500in PBS/0.05% TWEEN-20, 0.1 mL was added to each well, and plates wereincubated at 37° C. for two hours. Plates were washed (as above).Substrate solution was prepared by diluting p-nitrophenyl phosphate(Sigma) in diethanolamine at 1 mg/ml. Substrate was added to the plateat 0.1 mL per well and incubated at room temperature for one hour. Thereaction was stopped with 50 ul/well of 3N NaOH and plates were read at405 nm with 690 nm reference.

Recombinant PorA Induction: The BLR(DE3)/pET9a strains were grownovernight at 37° C. in HYSOY Broth (Sheffield Products) supplementedwith Kan-30 and 2% glucose. In the morning the 0/N cultures were diluted1/20 in HYSOY Broth Kan-30 and 1% glycerol and grown at 37° C. for 1hour. These cultures were induced by the addition of IPTG to a finalconcentration of 1 mM. The cultures were grown for an additional 2-3hours and then harvested.

Recombinant PorA Purification: The rPorA was solubilized from E. coliinclusion bodies with 8M Urea, and refolded by dialysis against buffercontaining no urea. The refolded rPorA was then concentrated bydiafiltration and buffer exchanged by G25 column into NaPO4 pH6. Thedialyzed rPorA was then run on a cation exchange column (S FRACTOGEL)and eluted with 1M NaCl.

The sOMPs from strain 8529 (P1.7-2.3) elicit PorA independentbactericidal activity in mice against strains expressing heterologousserosubtypes. The following table, Table IX, shows the bactericidalactivity in the studied strains.

TABLE IX Test Strain Serosubtype BC₅₀Titer¹ 539 P1.7-2,3 1280 539 PorA-NST² 1080 H44/76 P1.7,16 3285 H44/76 PorA- NST 2620 H355 P1.19,15 >1350H355 PorA- NST >1350 880049 P1.7-2,4 290 880049 PorA- NST 85 M982P1.22,9 85 M982 PorA- NST <50

Preparation of sOMPs: N. meningitidis membranes were extracted withTX-100, ZWITTERGENT 3-14, and ZWITTERGENT 3-14+0.5M NaCl. The sOMPsreferred to above were solubilized in the ZWITTERGENT 3-14/0.5M NaClextract. The extraction is performed using techniques well known topersons skilled in the art, for example, see U.S. Pat. No. 6,355,253which is hereby incorporated by reference.

Immunogencity: Female Swiss-Webster mice were immunized with 25 ug totalprotein adjuvanted with 20 ug QS-21 at week 0 and 4. An exsanguinationbleed and data analysis were done at week 6.

1 Bactericidal (BC₅₀) titers represented as the reciprocal of thedilution of anti-sera which reduces viable cell count by 50%. Week 0normal mouse sera had BC₅₀ titers of <25

2 NST=Non Serosubtypable

The following table, Table X, shows the purification andcharacterization summary for recombinant lipidated P2086 (rLP2086) forboth Subfamily A and Subfamily B.

Subfamily A rLP2086 Purification

TABLE X rLP2086 Variant A.A. Homology (%)¹ Theoretical pI Purity (%)²870446 75 6.1 80 2996 71 5.9 95 M97 252988 71 6.3 96 C11 68 6.4 82 M98250771 62 6.1 83Subfamily B rLP2086 Purification

TABLE XI rLP2086 Variant A.A. Homology (%)¹ Theoretical pI Purity (%)²8529 100 7.5 96 M982 94 6.3 96 88049 92 6.2 90 CDC1573 87 5.6 93

Purification Method: All variants were solubilized from E. colimembranes with TX-100 (exception rLP2086-8529 which was solubilized withSarcosyl or Urea). Further purification was achieved with a combinationof anion exchange (TMAE), size exclusion and/or cation exchange (SFRACTOGEL) chromatography in a Tris-HCl or NaPO4 buffer.

-   -   1 Amino acid homology as compared to P2086 from strain 8529    -   2 Purity as determined by SDS-PAGE and laser densitometry of        colloidal COOMASSIE stained band (SIMPLY BLUE stain)

Immunogenicity of a Subfamily B member, rLP2086-8529, tested againsthomologous and heterologous strains

Table XII below shows immunogenicity of a Subfamily B member,rLP2086-8529, tested against homologous and heterologous strains

TABLE XII P2086 Target A.A. Whole Cell Target Sub- Strain Homol-ELISA^(b) BC₅₀ Strain family Serosubtype ogy^(a) Titer Titer^(C) 539 BP1.7-2,3 100 >1,458,000 3,200 H44/76 B P1.7,16 100 >1,458,000 3,200 H355B P1.19,15 100 >1,458,000 3,200 CDC937 B P1.7-2,3-4 100 >1,458,000 >800M97 252097 B P1.7-2,16 100 >1,458,000 >800 870227 B P1.5-2,10100 >1,458,000 <25 6940 B P1.18,25,6 97 900,162 >800 M982 B P1.22,9 94435,909 200 880049 B P1.7-2,4 92 349,912 400 CDC1573 B P1.7-1,1 87102,508 25 870446 A P1.12-1,13 71 389,829 800 M98 250771 A P1.22,14 62139,397 <25 NmB A P1.5-1,2-2 71 <2,000 <25

Vaccination Procedure: 6-8 week old female Swiss-Webster mice wereimmunized with bug rLP2086-8529+20 ug QS-21 at week 0 and week 4. Dataanalysis was performed on the week 6 exsanguination bleed.

-   -   a Amino acid homology of P2086 as compared to rLP2086-8529    -   b Endpoint titers expressed as the reciprocal of the dilution at        absorbance=0.1    -   c BC50 titers represented as the reciprocal of the dilution of        anti-sera which reduces viable cell count by 50%. Week 0 normal        mouse sera had BC50 titers of <10

Table XIII shows immunogenicity of a Subfamily B member, rLP2086-2996,tested against homologous and heterologous strains.

TABLE XIII P2086 Target A.A. Whole Cell Target Sub- Strain Homol-ELISA^(b) BC₅₀ Strain family Serosubtype ogy^(a) Titer Titer^(C) NmB AP1.5-1,2-2 99.6 8,979 <25 870446 A P1.12-1,13 99 <1,458,000 >800 M97252697 A P1.18,25,6 98 320,732 >800 6557 A P1.22-1,14-1 98 17,319 <25M98 250732 A P1.22,14-1 89 241,510 >800 M98 250771 A P1.22,14 89 447,867800 H44/76 B P1.7,16 72 56,386 <25

Vaccination Procedure: 6-8 week old female Swiss-Webster mice wereimmunized with bug rLP2086-2996+20 ug QS-21 at week 0 and week 4. Dataanalysis was performed on the week 6 exsanguination bleed.

-   -   a Amino acid homology of P2086 as compared to rLP2086-2996    -   b Endpoint titers expressed as the reciprocal of the dilution at        absorbance=0.1    -   c Bactericidal (BC50) titers represented as the reciprocal of        the dilution of anti-sera which reduces viable cell count by        50%. Week 0 normal mouse sera had BC50 titers of <10.

Table XIV below shows that antisera to rLP2086 and rPorA arecomplimentary when mixed and assayed for bactericidal activity.

TABLE XIV H44/76 NMB 880049 H355 870227 6557 Antisera (P1.7,16)(P1.5-1,2-2) (P1.7-2,4) (P1.19,15) (P1.5-2,10) (P1.22-1,14-1)Anti-rLP2086 + >3,200 >800 200 >800 200 200 three rPorA antiseraControls anti-rLP2086 6,400 <25 100 3,200 <25 <25 Corresponding — 1,600— — 200 400 monovalent rPorA antisera

Vaccination Procedure: 6-8 week old female Swiss-Webster mice wereimmunized with either 10 ug rLP2086-8529/20 ug QS-21, or 15 ug rPorA/100ug MPL at week 0 and week 4. Data analysis was performed on the week 6exsanguination bleed.

-   -   a Bactericidal (BC50) titers represented as the reciprocal of        the dilution of anti-sera which reduces viable cell count by        50%. Week 0 normal mouse sera had BC50 titers of <10.

The following table, Table XV, shows that mixtures of rLP2086Subfamilies and two rPorAs elicit bactericidal antibody in mice.

TABLE XV M98 M98 M97 6940 880049 M98 250771 250732 252697 870446 NmB6557 H44/76 SfB SfB SfB SfA^(b) SfA SfA SfA SfA SfA SfB^(b) P1.18 P1.7-P1.22, P1.22, P1.22, P1.18, P1.12- P1.5- P1.22- P1.7,16 25,6 2,4 9 1414-1 25,6 1,13 1,2-2 1,14-1 Antigen rLP2086- >800 >800 200 400800 >800 >800 >800 — <25 8529 + rLP2086- 2996 rLP2086- >800 800 100 200400 400 >800 >800 >800 200 8529 + rLP2086- 2996 + rP1.5-1,2-2 + rP 1.22-1,14-1 Monovalent >800 >800 200 400 800 >800 >800 >800 >800 800Controls^(c)

Vaccination Procedure: 6-8 week old female Swiss-Webster mice wereimmunized with bug of each protein+20 ug QS-21 at week 0 and week 4.Data analysis was performed on the week 6 exsanguination bleed.

-   -   a Bactericidal (BC50) titers represented as the reciprocal of        the dilution of anti-sera which reduces viable cell count by        50%. Week 0 normal mouse sera had BC50 titers of <10.    -   b SfA—Subfamily A, SfB—Subfamily B    -   c Relevant monovalent control: rLP2086-8529, rLP2086-2996,        rP1.5-1.2-2 or rP1.22-1.14-1 antisera

The following summarizes the results of the above described studies.Anti-rLP2086 antisera is bactericidal against 13/16 test strains. Elevenstrains expressing different serosubtypes are killed by anti-P2086 sera.Bactericidal activity of anti-rLP2086 sera is complimentary toanti-rPorA sera. Mixtures of P2086 and PorA elicit complimentarybactericidal antibodies in mice. Differential detergent extraction,purification and immunization in conjunction with a functional antibodyassay against many strains can be used to identify new vaccinecandidates. P2086 has been identified as a vaccine candidate whichelicits bactericidal antibody against strains heterologous in both P2086and rPorA. Thus, the 2086 family of proteins may be a useful vaccineeither alone or in combination with other neisserial antigens.

Example 9

In accordance with the previous examples, additional meningococcalstrains, of varying serogroups, were screened by PCR for the presence ofthe ORF 2086 gene. Ultimately, one hundred meningococcal strains werescreened. The following describes the study and its overall results.These results supplement the data from the previous examples.

Two sets of internal PCR primers specific to the C-terminal variableregions were utilized to discriminate between Subfamily A and B genesequences. The presence of a PCR amplified product of approximately 350bp indicated that the 2086 gene sequence was present on the chromosome.All strains yielded a single PCR product of the expected size. Thenucleotide sequences of fifty-five full-length ORF 2086 genes weredetermined, aligned (DNAStar MegAlign) and used to generate aphylogenetic tree. (See FIG. 12).

Nine of these 2086 genes were recombinantly expressed as a rLP2086lipoprotein in a pBAD arabinose inducible promoter system and three ofthese genes were recombinantly expressed as a rP2086 non-lipidatedprotein in an IPTG inducible pET system. These recombinant proteins wereexpressed in E. coli B. The purified recombinant protein was used toimmunize mice and the mouse antisera was assayed for its serum IgGtiters and its bactericidal activity against a variety of heterologousmeningococcal strains.

ORF 2086 was amplified by PCR from one of the following, wholemeningococcal cells, purified chromosomal DNA or plasmid DNA templates.

Nine ORF 2086 genes were cloned into the vector pLP339, which fuses theHaemophilus P4 leader sequence to the 5′ end of the ORF 2086 genes. E.coli strain BLR was used as the host strain for recombinant expressionof the lipidated form of rP2086 from the pBAD/ORF 2086 clones. (See FIG.10A) The pBAD arabinose inducible promoter drives the expression the P4signal/ORF 2086 fusion protein to express a lipidated form of rP2086.Three P2086 genes, lacking a signal sequence, were cloned into a pET9avector behind the highly active T7 phage promoter. E. coli strainBL21(DE3) was used as the host strain for recombinant expression of anon-lipidated form of ORF 2086 from the pET9a/ORF 2086 clones. (See FIG.10B) The DE3 lysogen in E. coli strain BL21 can be induced to expressthe T7 RNA polymerase under the control of the lacUV5 promoter byaddition of IPTG. See, WCE; FEMS Micro. Lett., 48 (1987) 367-371 andBCA; J. Clin. Microbiol., 38 (2000) 2878-2884.

The gene, ORF2086, was cloned and sequenced from fifty-five different N.meningitidis strains. The nucleotide sequences were aligned (DNAStarMegAlign) and used to generate a phylogenetic tree. (See FIG. 12). Thistree reveals two distinct subfamilies of the ORF 2086 gene nucleotidesequence. The two subfamilies of genes are similar at their 5′ ends, butcontain considerable variation near their 3′ ends. Although thereappears to be significant variability, certain key regions of the geneare highly homologous amongst the different strains. These conservedregions may provide functional continuity for the protein and may beindicative of cross-protective epitopes to be exploited as vaccinetargets.

The 2086 gene was cloned from several serogroup B meningococcal strainsand expressed with and without the lipidation signal sequence. Referringto FIGS. 11A and 11B, gel photographs show the whole cell lysates of E.coli B expressing the r2086 protein. The non-lipidated form fused to theT7-Tag expressed at the highest level. The T7-Tag sequence may providestability to the mRNA and significantly enhances the level ofpolypeptide translated. This fusion protein appears to deposit ininclusion bodies and can be purified and refolded readily with knownprotocols. The lipidated and non-lipidated forms of P2086 are expressedat approximately 5 to 8% of total cellular protein, with the exceptionof the T7-Tag fusions, which express rP2086 as approximately 50% oftotal protein. The non-lipidated form of the protein appears to besoluble and localized in the cytoplasm. The lipidated form of theprotein appears to be associated with the membrane fractions and issolubilized with detergent.

The recombinant lipidated 2086 protein from N. meningitidis B strain8529 consistently elicits greater serum IgG titers than thenon-lipidated form (see Table XVI below), which correlates well with theenhanced level of bactericidal activity against both homologous andheterologous meningococcal strains (see Table XVII below). The proteinin its native lipidated form may have superior tertiary structure forantigen presentation and/or the attached lipid may act as an adjuvantstimulating a greater immunogenic response.

TABLE XVI Immune Response Elicited at Week 6 by WCE using 8529 rP2086(non-lipidated) vs. 8529 rLP2086 (lipidated) Mouse Sera Anti- Adju- genvant Meningococcal Strains (10 μg) (20 μg) H44/76 H355 870227 880049870446 rP2088 QS-21 273,238 212,947 102,694 69,124 21,466 rLP2086 QS-215,384,306 4,819,061 2,930,946 1,307,091 886,056

TABLE XVII 8529 rP2086 Elicits Weaker Bactericidal Activity than 8529rLP2086 Mouse Sera Antigen Adjuvant Meningococcal Strains (10 ug) (20ug) H44/76 H355 880049 NMB rP2086 QS-21 200 100 <25 <25 rLP2086 QS-216,400 3,200 100 <25 Pre- — <10 <10 <10 <10 Immune Positive — 1,600 100200 1,600 ControlThe following is a summary of the results of the study. All N.meningitidis B strains tested appear to have one 2086-like gene. Atleast two families of the 2086 gene are represented: Subfamily A—about30% of strains and Subfamily B—about 70% of strains. The 2086 gene hasbeen cloned and sequenced from 55 N. meningitidis strains. Sequenceswithin Subfamily A are ˜86-100% identical at the DNA level. Sequencewithin Subfamily B are ˜89.5-100% identical at the DNA level. Sequenceswithin Subfamily A vs. Subfamily B ˜60.9%-74% identical at the DNAlevel. 2086 homologs have been identified by PCR screening in thefollowing:

-   N. meningitidis A, B, C, W135, Y-   N. lactamica-   N. gonorrhoeae FA1090    Several ORF 2086 genes have been cloned and recombinantly expressed    -   Lipidated versions of P2086 were expressed from nine        meningococcal strains.    -   These recombinant proteins have been purified and used to        vaccinate mice.    -   The resulting antisera is bactericidal.

Non-lipidated versions of P2086 were expressed from three of the abovenine strains. rLP2086 consistently elicits a greater immune responsethan rP2086.

rLP2086 also exhibits enhanced bactericidal activity against bothhomologous and heterologous meningococcal strains.

Example 10

The following tables, Tables XVIII and XIX, show the characterization ofvariants of members of the two subfamilies.

TABLE XVIII Subfamily A rLP2086 Variants - Characterization rLP2086-rLP2086- rLP2086- rLP2086- rLP2086- 252988 250771 870446 2996 C11 GrowthHYSOY HYSOY HYSOY HYSOY HYSOY Media Solubility rTX-100 

TX-100 TX-100 rTX-100 

rTX-100 

Z3-12 Z3-12 Z3-12 Purification TMAE HQ Poros HQ Poros TMAE TMAE Steps SSEC SEC SEC S FRACTOGEL FRACTOGEL SEC Purity (%) 96 83 80 95 82 Yield0.2 0.7 0.8 0.5 0.1 (mg/g cell (fermentor) pellet) Size SEC 134,000155,000 132,000 163,000 126,000 (Z3-12) MS 27,897 — — 27,878 28,139 (712lipid) (750 lipid) (682 lipid) Thermal 66° C. — NT 65° C. 63° C.Denaturation Transition Midpoint (T_(M)) ° C. Protein 2.7 mg 1 mg(Z3-12) 5.0 mg 44 mg 1.1 mg Available (mg) 8529 71 62 71 72 68 SequenceHomology (%)

TABLE XIX Subfamily B rLP2086 Variants - Characterization rLP2086-rLP2086- rLP2086- rLP2086- 8529 M982 880049 CDC1573 Growth Media ApollonApollon HYSOY HYSOY (Sanford) Solubility 4M Urea 

rTX-100 

rTX-100 

rTX-100 Z3-12 Z3-12 Z3-12 Purification Steps TMAE TMAE TMAE TMAE SFRACTOGEL S FRACTOGEL S FRACTOGEL SEC Purity (%) 96 96 90 93 Yield 0.21.6 0.4 1.0 (mg/g cell pellet) (fermentor) (fermentor) Size SEC (Z3-12)95,000 110,000 100,000 120,000 150,000 MS 27,785 27,719 28,044 28,385(822 lipid) (711 lipid) (819 lipid) (823 lipid) Thermal Denaturation 70°C. 75° C. 62° C. NT Transition Midpoint (T_(M)) ° C. Protein Available(mg) Urea—34 mg Pool 1—47 mg 3.6 mg 4.9 mg Sarc—36 mg Pool 2—17 mg 8529Sequence 100 94 92 87 Homology (%)

Table XX below provides the results of fluorescent serum bactericidalassays for the 2086 Subfamily A.

TABLE XX M98 M97 Description 250771 870446 6557 NMB 250732 252697rLP2086-252988, >800 >800 <25 — >800 >800 10 μg (99%)* (99%)* (99%)*(93%)* rLP2086-C11, 200 >880 <25 — 200 400 10 μg (91%)*rLP2086-250771, >800 >800 <25 — >800 >800 10 μg (92%)* (99%)* (96%)*(84%)* rLP2086-870446, 400 >800 <25 — 400 400 10 μg (99%)* rLP2086-2996,800 >800 <25 — >800 >800 10 μg (99%)* (93%)* (72%)* rLP2086-8529 +800 >800 <25 — >800 >800 rLP2086-2996, (99%)* (80%)* (72%)* 10 μgrLP2086-8529 + — 800 200 >800 — — rP1.22a, 14a + (98%)* rP1.5a, 2c, 10μg rLP2086-8529 + 400 >800 200 >800 400 >800 rLP2086-2996 + (99%)*(99%)* (88%)* rP1.22a, 14a + rP1.5a, 2c, 10 μg NMB/rLP2086- — 100 — 400— — 8529 vesicles, 20 μg rP1.22a, 14a, 10 μg 25 — 800 — 100 — rP1.5a,2c, 10 μg — — — >800 — — (99%)* rLP2086-8529, — 800 — — — — 10 μgrP1.22a, 14a, 25 μg 200 — — — 800 — rP1.18, 25.6, 5 μg — — — — — nP1.22,9 (M982), — — 100 — — — 25 μg pre-immune mouse <10 <10 <10 <10 <10 <10serum (negative control) 800 400 800 1600 ** ** Notes: *Percentageindicates the % BC activity at the 1:800 dilution. ** Positive controlnot available. — serum not tested

Example 11

The following further demonstrates that P2086 is expressed in neisserialstrains and provides additional specific examples of P2086 expression inseveral strains.

Cell lysates were prepared with cells from plate cultures resuspended inSDS sample buffer and heated at 98° C. for four minutes. Samples wereloaded at ˜30-50 ug total protein per well on 10-20% pre-cast gels (ICN)and run at 175V. The gels were transferred to a nitrocellulose membrane,which was then blocked for 30 min. with 5% powdered milk inTris-buffered saline (BLOTTO). The primary antibody used was a pool ofpolyclonal antisera raised against individual rLP2086 variants in mice.

Referring to FIGS. 17 and 18, a Western Blot shows the reactivity ofrLP2086 mouse antisera to P2086 Subfamily A and B whole cell lysates.For the Subfamily A cell lysate blot, the antisera used were raisedagainst rLP2086-2996, -870446 and -250771 with rLP2086-250771 diluted at1/500 in BLOTTO and the others diluted at 1/1000 in BLOTTO. For theSubfamily B cell lysate blot, the antisera used were raised againstrLP2086-8529 (diluted 1/1000 in BLOTTO), -CDC1573. -M982 and -880049(these three diluted 1/500 in BLOTTO). The primary antisera and blotwere incubated at 4° C. overnight. The blot was washed, agoat-anti-mouseAP secondary was added at 1/500 in BLOTTO, and the blotwas incubated for 30 min. at room temperature. After washing, the blotwas developed using the BCIP/NBT Membrane Phosphatase Substrate System(KPL).

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The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein. The foregoing describes the preferred embodimentsof the present invention along with a number of possible alternatives.These embodiments, however, are merely for example and the invention isnot restricted thereto.

What is claimed is:
 1. A composition comprising an isolated protein having the amino acid sequence set forth in SEQ ID NO:
 250. 2. The composition of claim 1, wherein the protein is nonpathogenic.
 3. The composition of claim 1, wherein the protein is lipidated.
 4. The composition of claim 1, wherein the protein is isolated from a Neisseria bacterium.
 5. The composition of claim 4, wherein the bacterium is Neisseria meningitidis.
 6. The composition of claim 1, further comprising a polysaccharide.
 7. The composition of claim 1, further comprising an adjuvant.
 8. The composition of claim 7, wherein the adjuvant is aluminum phosphate.
 9. The composition of claim 1, further comprising a buffer.
 10. The composition of claim 1, wherein the protein is encoded by the sequence set forth in SEQ ID NO:
 251. 